Methods for in vitro differentiation of TH-17+ cells

ABSTRACT

The present invention is directed to an in vitro method for promoting differentiation and proliferation of human T helper lymphocytes that express IL17 (Th-IL17+ cells). The instant method may be used to generate a population of human T helper lymphocytes that express IL17 (Th-IL17+ cells) in vitro. Methods for screening to identify agents capable of modulating Th-IL17+ cell differentiation are also encompassed by the present invention. Isolated, pure populations of homogeneous Th-IL17+ cells that do not express cellular markers characteristic of Th1, Th2, or Treg cells are also encompassed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional Application of co-pending U.S.application Ser. No. 13/473,980, filed May 17, 2012, which in turnclaims priority to U.S. application Ser. No. 12/386,355, filed Apr. 15,2009, now U.S. Pat. No. 8,183,040, issued May 22, 2012, which in turnclaims priority under 35 USC §119(e) from U.S. Provisional ApplicationSer. No. 61/124,242, filed Apr. 15, 2008, each of which applications isherein specifically incorporated by reference in its entirety.

The research leading to the present invention was funded in part by NIHgrants 5 R37 AI033303 and R01 AI065303. The United States government mayhave certain rights in the invention.

FIELD OF THE INVENTION

The present invention pertains to the fields of cell culture,immunology, and T lymphocytes. More specifically, the invention relatesto in vitro methods directed to promoting differentiation andproliferation of human Th-17+ cells and to in vitro screening methodsdirected to identifying agents capable of modulating human Th-17+ cellsdifferentiation. Homogenous populations of Th-17+ cells generated usingthe methods of the invention are also described herein.

BACKGROUND OF THE INVENTION

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Th17 cells, the T helper cells that produce IL-17 and otherpro-inflammatory cytokines, have been shown to have key functions in awide variety of autoimmune disease models in mice and are thought to besimilarly involved in human disease (reviewed¹⁻³). In healthy humans,IL-17-secreting cells are present in the CD45RO⁺CCR6⁺ populations of Tcells from peripheral blood^(4,5) and gut⁵. Th17 cells or their productshave been associated with the pathology of multiple inflammatory orautoimmune disorders in humans. IL-17 protein and Th17 CD4⁺ T cells arefound in lesions from multiple sclerosis patients⁶⁻⁸ where they arethought to contribute to the disruption of the blood-brain barrier⁹.IL-17 is produced by CD4⁺ T cells of rheumatoid synovium¹⁰ and isthought to contribute to inflammation in rheumatoid arthritis^(11,12).In psoriasis, products associated with Th17 cells, including IL-17,IL-17F, IL-22, and CCR6 are induced¹³⁻¹⁵. IL-17 is induced in the gutmucosa from Crohn's disease and ulcerative colitis patients and Th17cells are detected^(13,16). IL-23, which is produced by dendritic cellsin the intestine¹⁷, contributes significantly to Th17 celldifferentiation¹⁸. Strikingly, polymorphisms in the IL23R gene areassociated with Crohn's disease, further implicating the Th17 cellpathway in the pathogenesis of this disease¹⁹.

The mechanisms leading to differentiation of Th17 cells have been wellestablished in mice but they are still poorly understood in humans. Inmice, differentiation of Th17 cells that secrete IL-17 (also referred toas IL-17A) and IL-17F requires the expression of the transcriptionfactors Rorγt, an orphan nuclear hormone receptor, STAT3 and IRF4(reviewed in²⁰). Rorγt is sufficient to direct expression of IL-17 inactivated mouse T cells²¹ and is thus considered to be the effectortranscription factor that establishes the Th17 differentiation lineage.Conditions that induce Th17 cell differentiation from naive murine Tcells, including expression of Rorγt, have been established.Combinations of TGF-β and IL-6²²⁻²⁴ or TGF-β and IL-21²⁵⁻²⁷ aresufficient to initiate IL-17 and IL-17F expression. Expression of IL-22,considered to be another Th17 cytokine, is induced by IL-6 and inhibitedby high concentrations of TGF-β¹⁴. IL-23 is required in vivo for thegeneration of pathogenic Th17 cells, but it is not required in vitro forthe induction of IL-17, IL-17F or IL-22¹⁸.

In contrast to murine T cells, human T cells with a naive surfacephenotype fail to produce IL-17 in the presence of TGF-β and IL-6²⁸⁻³¹.Increased expression of IL-17 was, however, observed by some groups inresponse to IL-1β alone²⁹ or with IL-23¹⁵. Others have failed to observesuch a response³⁰. These disparate findings reveal that the identitiesof the exogenous factors required to induce the differentiation of humanTh17 cells remain unknown. The difference between the requirements formouse and human Th17 cell differentiation have been ascribed todivergent differentiation processes, although it remains possible that Tcells purified from adult peripheral blood on the basis of CD45RAexpression alone are not equivalent to naive murine T cells³²⁻³⁴.

SUMMARY OF INVENTION

Th17 cells are IL-17-secreting CD4⁺ T cells involved in autoimmunedisease and mucosal immunity. In naive CD4⁺ T cells from mice, IL-17 isexpressed in response to a combination of IL-6 or IL-21 and TGF-β andrequires induction of the transcription factor Rorγt. It has beensuggested that human Th17 cell differentiation is independent of TGF-βand thus differs fundamentally from mouse. The present inventorsdemonstrate here that, in serum-free conditions, a combination of TGF-βwith IL-1β and any one of IL-6, IL-21 or IL-23 is necessary andsufficient to induce IL-17 expression in naive human CD4⁺ T cellsisolated from cord blood.

In one aspect, the present invention is directed to a method forpromoting differentiation and proliferation of human T helperlymphocytes that express IL17 (Th-IL17+ cells) in vitro, the methodcomprising the steps of: isolating a population of naive CD4+ T cellsfrom a human; and incubating the population of naive CD4+ T cells inserum-free culture medium comprising TGF-β, IL-1β, and any one of IL-6,IL-21 or IL-23, wherein the incubating promotes differentiation of humanTh-IL17+ cells. Detecting an increase in expression of any cellularmarker of human Th-IL17+ differentiation, such as IL17, IL17F, IL23R,RORC or IL26, after incubating in human Th-IL17+ promoting conditionsmay be used as a positive indicator of Th-IL17+ cell differentiation.

In another aspect, the invention is directed to a method for generatinga population of human T helper lymphocytes that express IL17 (Th-IL17+cells) in vitro, the method comprising the steps of: isolating apopulation of naive CD4+ T cells from a human; and incubating thepopulation of naive CD4+ T cells in serum-free culture medium comprisingTGF-β, IL-1β, and any one of IL-6, IL-21 or IL-23, wherein theincubating promotes differentiation and proliferation of human Th-IL17+cells and thereby generates a population of human Th-IL17+ cells.

The naive CD4+ T cells used in the present method may be isolated fromcord blood, buffy coats of adult humans, cell cultures comprising cellsthat express CD34 (CD34+ cells), or human embryonic stem cells. CD34+cells may be isolated from fetal liver, cord blood, or mobilized adultblood and further expanded in vitro to generate cell cultures comprisingCD34+ cells.

In an embodiment of the invention, the concentration of TGF-β in theserum-free culture medium is at least 0.1 ng/ml. In a more particularembodiment, the concentration of TGF-β in the serum-free culture mediumis about 10 ng/ml or is 10 ng/ml.

In an embodiment of the invention, the concentration of IL-1β in theserum-free culture medium is at least 0.1 ng/ml. In a more particularembodiment, the concentration of IL-1β in the serum-free culture mediumis about 10 ng/ml or is 10 ng/ml.

In an embodiment of the invention, the concentration of IL-6 in theserum-free culture medium is at least 0.1 ng/ml. In a more particularembodiment, the concentration of IL-6 in the serum-free culture mediumis about 10 ng/ml or is 10 ng/ml.

In an embodiment of the invention, the concentration of IL-21 in theserum-free culture medium is at least 0.1 ng/ml. In a more particularembodiment, the concentration of IL-21 in the serum-free culture mediumis about 10 ng/ml or is 10 ng/ml.

In an embodiment of the invention, the concentration of IL-23 in theserum-free culture medium is at least 0.1 ng/ml. In a more particularembodiment, the concentration of IL-23 in the serum-free culture mediumis about 10 ng/ml or is 10 ng/ml.

In yet another embodiment of the invention, the serum-free culturemedium further comprises IL-2. More particularly, the concentration ofIL-2 in the serum-free culture medium is at least 1 U/ml. Even moreparticularly, the concentration of IL-2 in the serum-free culture mediumis about 10 U/ml or is 10 U/ml.

In an aspect of the invention, the population of CD4+ T cells isactivated with anti-CD3 and anti-CD28 antibodies prior to or concomitantwith the incubating step.

In another aspect of the invention, the incubating step is at leastthree days. More particularly, the incubating step is about six days oris six days.

The invention also encompasses a method wherein the population of CD4+ Tcells is transduced with a lentiviral vector comprising a nucleic acidsequence encoding an exogenous polypeptide RORγT before or during theincubating step. More particularly, the lentiviral vector comprises anucleic acid sequence encoding RORγT or RORγ. The nucleic and amino acidsequences of human and mouse RORγT and RORγ are presented herein. Asunderstood in the art, the gene RORC encodes RORγ and RORγT, which areisoform a and b, respectively, of RORC. In another embodiment of theinvention, the lentiviral vector comprises a nucleic acid sequenceencoding human RORα or human RORβ, which can also drive IL-17 expressionin human CD4+ cells. Nucleic acid sequences encoding human RORα or humanRORβ are presented herein.

The invention also encompasses a method wherein the population of CD4+ Tcells is isolated based on positive cell surface staining for cellsurface antigens or receptors. Exemplary cell surface antigens orreceptors include putative homing receptors: CCR7, CCR6, CCR5, CXCR4,CD62L, CD44, CD11a, CD27, CD57, and CD49f.

The invention also encompasses a method wherein the population of CD4+ Tcells is isolated based on positive cell surface staining for aparticular T cell receptor. In an embodiment of the invention, the Tcell receptor is specific for a particular tumor cell antigen.

The method of the present invention may further comprise an enrichmentstep, whereby post-incubation cells (i.e., cells that have beenincubated in accordance with the invention) are selected for expressionof a cell surface marker or cell surface antigen expressed on humanTh-IL17+ cells. More particularly, the cell surface marker or cellsurface antigen expressed on human Th-IL17+ cells is CCR6, CCR7, CCR5,and CXCR4.

The present invention also encompasses a method for screening toidentify an agent that modulates human Th-IL17+ cell differentiation invitro, the method comprising the steps of:

a) isolating a population of CD4+ T cells from a human and dividing thepopulation into at least a first and second sub-population of CD4+ Tcells;

b) incubating a first sub-population of CD4+ T cells in serum-freeculture medium comprising TGF-β, IL-1β, and any one of IL-6, IL-21 orIL-23, wherein the incubating promotes differentiation of human Th-IL17+cells;

c) incubating a second sub-population of CD4+ T cells in serum-freeculture medium comprising TGF-β, IL-1β, and any one of IL-6, IL-21 orIL-23, and an agent;

d) detecting expression of IL17, IL17F, IL23R, RORC or IL26 in each ofsaid first and second sub-populations of CD4+ T cells incubated withoutor with the agent;

e) comparing the expression of IL17, IL17F, IL23R, RORC, IL26, or FOXP3in each of said first and second sub-populations of CD4+ T cellsgenerated following incubation without or with the agent, wherein achange in expression of IL17, IL17F, IL23R, RORC, IL26, or FOXP3following incubation with the agent relative to the expression of IL17,IL17F, IL23R, RORC, IL26, or FOXP3 following incubation without theagent indicates that the agent is a modulator of human Th-IL17+ celldifferentiation in vitro.

In another embodiment of the invention, the serum-free culture medium ofthe screening method further comprises IL-2. More particularly, theconcentration of IL-2 in the serum-free culture medium is at least 1U/ml. Even more particularly, the concentration of IL-2 in theserum-free culture medium is about 10 U/ml or is 10 U/ml.

In an aspect of the screening method, the change in expression of IL17,IL17F, IL23R, RORC or IL26 is a decrease in expression of IL17, IL17F,IL23R, RORC or IL26 following incubation with the agent, wherein thedecrease indicates that the agent is an inhibitor of human Th-IL17+ celldifferentiation in vitro.

In another aspect of the screening method, the change in expression ofIL17, IL17F, IL23R, RORC or IL26 is an increase in expression of IL17,IL17F, IL23R, RORC or IL26 following incubation with the agent, whereinthe increase indicates that the agent is a promoter or inducer of humanTh-IL17+ cell differentiation in vitro.

In yet another aspect of the invention, expression of FOXP3 is used as atarget for readout of the methods described herein. FOXP3 is a negativeregulator of human Th-IL17+ cell differentiation. Thus, in a screeningmethod of the invention, a practitioner could screen for an inhibitor ofTh17 that could increase FOXP3 expression and an inducer of Th17 thatcould inhibit FOXP3 expression. Given the role of FOXP3 in autoimmunity,the identification of such inhibitors and inducers would providepromising therapeutics.

With respect to the screening method, therefore, a decrease inexpression of FOXP3 following incubation with an agent indicates thatthe agent is a promoter of human Th-IL17+ cell differentiation in vitro.In contrast, an increase in expression of FOXP3 following incubationwith an agent indicates that the agent is an inhibitor of human Th-IL17+cell differentiation in vitro.

As described herein, the agent used in the screening method of theinvention may be a small molecule; polypeptide; azole-containingcompounds; cholesterol derivative compounds; retinoid derivativecompounds; shRNA/siRNA; neutralizing/blocking antibodies; tryptophanderivative compounds; Vitamin D derivatives; or molecules known toinhibit of fever, inflammation, or regulatory T (Treg) celldifferentiation.

An agent tested in a screening method of the invention may be added tothe culture medium before, during, or after addition of TGF-β, IL-1β,and any one of IL-6, IL-21 or IL-23.

In an aspect of the screening method, expression of IL17, IL17F, IL23R,RORC or IL26 is determined by polymerase chain reaction amplificationusing primer pairs specific for IL17, IL17F, IL23R, RORC or IL26.

The present invention further encompasses an isolated homogeneouspopulation of human Th-IL17+ cells, wherein the isolated homogeneouspopulation comprises at least 1×10⁹ human Th-IL17+ cells, wherein thehuman Th-IL17+ cells do not express cellular markers of otherdifferentiated Th cells. In accordance with the present invention, theisolated homogeneous population of human Th-IL17+ cells generated fromnaive CD4+ cells do not express cellular markers characteristic of Th1(e.g., IFNγ), Th2 (e.g., IL4 or IL13), or Treg cells (e.g., FoxP3). Itis understood that cellular markers characteristic of Th1, Th2, or Tregcells would be present in any population of Th-IL17+ cells generatedfrom memory Th cells. The isolated population of human Th-IL17+ cellsgenerated from naive CD4+ cells as described herein is, therefore,homogeneous with respect to the expression of only those cellularmarkers characteristic of Th17+ cells. Exemplary markers of humanTh-IL17 cells are IL-17, IL-17F, IL-26, and IL23R.

Other features and advantages of the invention will be apparent from thefollowing description of the particular embodiments thereof, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a-f: RORγT is Necessary and Sufficient for the Expression ofIL-17 in Human CD4⁺ T Cells. (a) Sorted CD45RO⁻ and CD45RO⁺CCR6⁺ wereactivated and expanded in the presence of IL-2 with or without IL-1β.IL-17 and IFNγ production was analyzed at day 6. (b, c) SortedCD45RO⁺CCR6⁺ were transduced with an empty vector or vector encodingRORγT-specific shRNA (shRNA-1 and shRNA-2). Cells were selected inpuromycin at day 2. RORC and β-actin mRNA expression (b) and IL-17 andIFNγ production (c) were analyzed at day 6 (representative experiment,n=4). (d) Naive cord blood CD4⁺ T cells were activated, transduced byvectors encoding IRES-HSA or RORγT-IRES-HSA and expanded for 6 days inthe presence of IL-2. IL-17 and IFNγ production was analyzed at day 6.(e) Naive cord blood CD4⁺ T cells were transduced with vectors encodingIRES-GFP or RORγT-IRES-GFP. GFP⁺ cells were sorted at day 6 and mRNAlevels of β-actin, IL-17, IL-17F and IL-26 were analyzed. (f) Naive cordblood CD4⁺ T cells were transduced with vectors encoding IRES-HSA,RORγT-IRES-HSA, GATA-3-IRES-HSA or T-bet-IRES-HSA. CCR6 cell surfaceexpression was measured at day 12.

FIG. 2a-d : TGF-β Induces RORγT but Inhibits its Activity and thisInhibition is Relieved by Inflammatory Cytokines. (a) Naive cord bloodCD4⁺ T cells were transduced with a vector encoding RORγT-IRES-HSA aloneor with a combination of IL-1β, IL-6, IL-21 and increasingconcentrations of TGF-β, and IL-17 intracellular staining was performedat day 6. (b) RORC and β-actin expression was measured in freshly sortedCCR4^(−/+)CCR6^(−/+) adult memory CD4⁺ T cells and in naive CD4⁺ T cellscultivated for 3 days in the presence of various cytokines. (c) RORC andβ-actin expression was analyzed in naive cord blood CD4⁺ T cells thatwere cultivated in various concentrations of TGF-β. (d) Naive cord bloodCD4⁺ T cells were transduced with a vector encoding RORγT-IRES-HSA ineither RPMI-10% FBS, RPMI-10% with an anti-TGF-β antibody or serum-freemedia. IL-17 and FOXP3 expression were analyzed at day 6. Arepresentative donor with low IL-17 expression following RORγTtransduction in the presence of serum is shown.

FIG. 3a-f : TGF-β, IL-1β and Either IL-6, IL-21 or IL-23 are Requiredfor Human Th17 Cell Polarization in Serum Free Conditions. (a) Naivecord blood CD4⁺ T cells were activated without cytokines or with IL-1β,IL-6 or IL-21 with or without IL-23, alone or with increasingconcentrations of TGF-β. IL-2 was added at day 3 and IL-17 expressionwas analyzed at day 14. (b) Naive cord blood CD4⁺ T cells were activatedwith no cytokine or with a combination of IL-1β+IL-23+TGF-β (1 ng/ml),with or without 10 U/ml IL-2. IL-17 expression was analyzed at day 6.(c) Naive cord blood CD4⁺ T cells were cultivated with IL-23+IL-2 aloneor together with IL-1β, IL-21 or IL-23 and increasing concentrations ofTGF-β. IL-17 and IFNγ expression was analyzed at day 6. (d) Time-courseof IL-17 and IFNγ production in naive cord blood CD4⁺ T cells polarizedin the presence of IL-2+IL-1β+IL-23+TGF-β (0.1, 1 or 10 ng/ml). (e)Summary of IL-17 expression in day 6 cultures of naive cord blood CD4⁺ Tcells from different donors (n=11) in the presence ofIL-2+IL-1β+IL-23+10 ng/ml TGF-β. (f) Naive cord blood CD4⁺ T cells werecultivated in IL-2 alone or IL-23+IL-1β+IL-2 with increasingconcentrations of TGF-β. IL-17 and IL-22 expression was analyzed at day6. Each panel is representative of at least three independent donors.

FIG. 4a-e : Induction of IL26, IL17F, IL17A, RORC and IL23R mRNA DuringHuman Th17 Cell Differentiation. (a, b, c) Naive cord blood CD4⁺ T cellswere cultivated with IL-2 alone or with IL-1β+IL-2, IL-23+IL-2, orIL-23+IL-1β+IL-2 with increasing concentrations of TGF-β. mRNA levels ofβ-actin, IL17 (a), IL26 (b) and IL17F (c) were analyzed on day 6following restimulation with PMA and ionomycin. (d, e) Naive cord bloodCD4⁺ T cells were cultivated with IL-2 alone or with IL-1β+IL-2,IL-23+IL-2 or IL-23+IL-1β+IL-2 with increasing concentrations of TGF-β.mRNA levels of β-actin, RORC (d) and IL23R (e) were analyzed on day 6.Each panel is representative of three independent donors.

FIG. 5a-c : Expression of CCR6 and FOXP3 During Human Th17 CellDifferentiation. (a) Naive cord blood CD4⁺ T cells were cultivated withIL-2 alone or with IL-1β+IL-2, IL-23+IL-2, or IL-23+IL-1β+IL-2 withincreasing concentrations of TGF-β. CCR6 expression was analyzed bysurface staining at day 6. (b) Naive cord blood CD4⁺ T cells werecultivated for 6 days in IL-2+IL-23+IL-1β+IL-2+10 ng/ml TGF-β. CCR6⁺ andCCR6⁻ cells were sorted and IL-17 was analyzed. (c) Naive cord bloodCD4⁺ T cells were cultivated with IL-2 alone or with IL-1β+IL-2,IL-23+IL-2, or IL-23+IL-1β+IL-2 with increasing concentrations of TGF-β.FOXP3 and IL-17 expression were analyzed at day 6.

FIG. 6: IL-17 Expression Following RORγT, RORαd and RORβ Transduction.Naive cord blood CD4⁺ T cells were transduced with RORγT, RORαd and RORβin RPMI-10% FBS alone. IL-17 was analyzed at day 6.

FIG. 7a-b : IL26 Gene has been Lost in Mice and Rats. (a) Genomicorganization of the IL22 locus based on UCSC genome browser. In humans,IL22 is followed by IL26 and IFNG in the same locus. In the mouse, Il22is followed by Iltifb (a duplication of Il22) and Ifng, and Il26 isabsent. (b) Among species, IL26 can be detected in pre-placentalvertebrates, but cannot be found in the mouse or rat genome. GenBankaccession numbers and amino acid identity to human IL-26 are indicated.Human IL-22 is shown for comparison.

FIG. 8: TGF-β Anti-TGF-β Effect on IL-17 Expression Following RORγTTransduction. Naive cord blood CD4⁺ T cells were transduced with RORγTin RPMI-10% FBS alone, with recombinant TGF-β or with an anti-TGF-βantibody. IL-17 and IFNγ expression was analyzed at day 6 in GFP^(hi)cells.

FIG. 9a-c : Expression and Neutralization of IL-6 and IL-21. (a, b)Naive cord blood CD4⁺ T cells were cultivated in IL-2 alone or,IL-1β+IL-2, IL-23+IL-2, or IL-23+IL-1β+IL-2 with increasingconcentrations of TGF-β. mRNA levels of β-actin, IL6 (a) and IL21 (b)was analyzed on day 6 following restimulation with PMA and ionomycin.(c) Naive cord blood CD4⁺ T cells were cultivated in IL-2 alone or,IL-23+IL-1β+IL-2 with increasing concentrations of TGF-β. Increasingconcentrations of neutralizing anti-IL-6 antibody and soluble IL-21receptor were also added. IL-17 expression was analyzed at day 6.

FIG. 10: Retinoic Acid Inhibits IL-17 Induction. Naive cord blood CD4⁺ Tcells were activated with a combination of IL-2+IL-1β+IL-23+TGF-β (0.1,1 and 10 ng/ml), with or without retinoic acid (10 and 100 nM). IL-17and IFNγ expression was analyzed at day 6.

FIG. 11a-b : Expression of RORA. (a) Naive cord blood CD4⁺ T cells werecultivated in IL-2 alone or, IL-1β+IL-2, IL-23+IL-2, or IL-23+IL-1β+IL-2with increasing concentrations of TGF-β. mRNA levels of β-actin and RORAwere analyzed on day 6. (b) RORA and β-actin expression was measured infreshly sorted CCR4^(−/+)CCR6^(−/+) adult memory CD4⁺ T cells.

DETAILED DESCRIPTION OF THE INVENTION

In order to more clearly set forth the parameters of the presentinvention, the following definitions are used:

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, reference to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

The term “complementary” refers to two DNA strands that exhibitsubstantial normal base pairing characteristics. Complementary DNA may,however, contain one or more mismatches.

The term “hybridization” refers to the hydrogen bonding that occursbetween two complementary DNA strands.

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single or double stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form. In discussing nucleic acid molecules, a sequence orstructure of a particular nucleic acid molecule may be described hereinaccording to the normal convention of providing the sequence in the 5′to 3′ direction.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous in the naturally occurring genome of the organismin which it originated. For example, an “isolated nucleic acid” maycomprise a DNA molecule inserted into a vector, such as a plasmid orvirus vector, or integrated into the genomic DNA of a prokaryotic oreukaryotic cell or host organism.

When applied to RNA, the term “isolated nucleic acid” refers primarilyto an RNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it isgenerally associated in its natural state (i.e., in cells or tissues).An isolated nucleic acid (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

The term “functional” as used herein implies that the nucleic or aminoacid sequence is functional for the recited assay or purpose.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID No:. For example, when used in reference to an amino acidsequence, the phrase includes the sequence per se and molecularmodifications that would not affect the basic and novel characteristicsof the sequence.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, phage or virus, that is capable of replication largely under itsown control. A replicon may be either RNA or DNA and may be single ordouble stranded.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage orvirus, to which another genetic sequence or element (either DNA or RNA)may be attached so as to bring about the replication of the attachedsequence or element.

An “expression vector” or “expression operon” refers to a nucleic acidsegment that may possess transcriptional and translational controlsequences, such as promoters, enhancers, translational start signals(e.g., ATG or AUG codons), polyadenylation signals, terminators, and thelike, and which facilitate the expression of a polypeptide codingsequence in a host cell or organism.

As used herein, the term “operably linked” refers to a regulatorysequence capable of mediating the expression of a coding sequence andwhich is placed in a DNA molecule (e.g., an expression vector) in anappropriate position relative to the coding sequence so as to effectexpression of the coding sequence. This same definition is sometimesapplied to the arrangement of coding sequences and transcription controlelements (e.g. promoters, enhancers, and termination elements) in anexpression vector. This definition is also sometimes applied to thearrangement of nucleic acid sequences of a first and a second nucleicacid molecule wherein a hybrid nucleic acid molecule is generated.

The term “oligonucleotide,” as used herein refers to primers and probesof the present invention, and is defined as a nucleic acid moleculecomprised of two or more ribo- or deoxyribonucleotides, preferably morethan three. The exact size of the oligonucleotide will depend on variousfactors and on the particular application and use of theoligonucleotide.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be “substantially” complementary to differentstrands of a particular target nucleic acid sequence. This means thatthe probes must be sufficiently complementary so as to be able to“specifically hybridize” or anneal with their respective target strandsunder a set of pre-determined conditions. Therefore, the probe sequenceneed not reflect the exact complementary sequence of the target. Forexample, a non-complementary nucleotide fragment may be attached to the5′ or 3′ end of the probe, with the remainder of the probe sequencebeing complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecifically.

The term “specifically hybridize” refers to the association between twosingle-stranded nucleic acid molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention, to thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

Primers may be labeled fluorescently with 6-carboxyfluorescein (6-FAM).Alternatively primers may be labeled with4,7,2′,7′-Tetrachloro-6-carboxyfluorescein (TET). Other alternative DNAlabeling methods are known in the art and are contemplated to be withinthe scope of the invention.

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into, for example, immunogenic preparations orpharmaceutically acceptable preparations.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More particularly, the preparationcomprises at least 75% by weight, and most particularly 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like). “Mature protein” or“mature polypeptide” shall mean a polypeptide possessing the sequence ofthe polypeptide after any processing events that normally occur to thepolypeptide during the course of its genesis, such as proteolyticprocessing from a polypeptide precursor. In designating the sequence orboundaries of a mature protein, the first amino acid of the matureprotein sequence is designated as amino acid residue 1.

The term “tag”, “tag sequence” or “protein tag” refers to a chemicalmoiety, either a nucleotide, oligonucleotide, polynucleotide or an aminoacid, peptide or protein or other chemical, that when added to anothersequence, provides additional utility or confers useful properties tothe sequence, particularly with regard to methods relating to thedetection or isolation of the sequence. Thus, for example, a homopolymernucleic acid sequence or a nucleic acid sequence complementary to acapture oligonucleotide may be added to a primer or probe sequence tofacilitate the subsequent isolation of an extension product orhybridized product. In the case of protein tags, histidine residues(e.g., 4 to 8 consecutive histidine residues) may be added to either theamino- or carboxy-terminus of a protein to facilitate protein isolationby chelating metal chromatography. Alternatively, amino acid sequences,peptides, proteins or fusion partners representing epitopes or bindingdeterminants reactive with specific antibody molecules or othermolecules (e.g., flag epitope, c-myc epitope, transmembrane epitope ofthe influenza A virus hemaglutinin protein, protein A, cellulose bindingdomain, calmodulin binding protein, maltose binding protein, chitinbinding domain, glutathione S-transferase, and the like) may be added toproteins to facilitate protein isolation by procedures such as affinityor immunoaffinity chromatography. Chemical tag moieties include suchmolecules as biotin, which may be added to either nucleic acids orproteins and facilitates isolation or detection by interaction withavidin reagents, and the like. Numerous other tag moieties are known to,and can be envisioned by, the trained artisan, and are contemplated tobe within the scope of this definition.

The terms “transform”, “transfect”, “transduce”, shall refer to anymethod or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, viraltransduction, transfection, electroporation, microinjection, PEG-fusionand the like.

The introduced nucleic acid may or may not be integrated (covalentlylinked) into nucleic acid of the recipient cell or organism. Inbacterial, yeast, plant and mammalian cells, for example, the introducednucleic acid may be maintained as an episomal element or independentreplicon such as a plasmid. Alternatively, the introduced nucleic acidmay become integrated into the nucleic acid of the recipient cell ororganism and be stably maintained in that cell or organism and furtherpassed on or inherited to progeny cells or organisms of the recipientcell or organism. In other applications, the introduced nucleic acid mayexist in the recipient cell or host organism only transiently.

A “clone” or “clonal cell population” is a population of cells derivedfrom a single cell or common ancestor by mitosis.

A “cell line” is a clone of a primary cell or cell population that iscapable of stable growth in vitro for many generations.

An “immune response” signifies any reaction produced by an antigen, suchas a protein antigen, in a host having a functioning immune system.Immune responses may be either humoral, involving production ofimmunoglobulins or antibodies, or cellular, involving various types of Band T lymphocytes, dendritic cells, macrophages, antigen presentingcells and the like, or both. Immune responses may also involve theproduction or elaboration of various effector molecules such ascytokines, lymphokines and the like. Immune responses may be measuredboth in in vitro and in various cellular or animal systems.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof, that binds to a specific antigen. Theterm includes polyclonal, monoclonal, chimeric, and bispecificantibodies. As used herein, antibody or antibody molecule contemplatesboth an intact immunoglobulin molecule and an immunologically activeportion of an immunoglobulin molecule such as those portions known inthe art as Fab, Fab′, F(ab′)2 and F(v).

The term “about” as used herein refers to a variation in a stated valueor indicated amount of up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.75%, 0.5%, 0.25% or 0.1%, wherein the variation can be either anincrease or a decrease in the stated value or indicated amount. Use ofthe term may, therefore, be used to establish a range of values oramounts.

As used herein, the term “naive CD4+ T cells” refers to a CD4+ T cellthat is functionally defined by the expression of cell surface markersof naivety that include CD45RA+CD25-HLA-DR−.

As used herein, the term “serum-free culture medium” is defined asserum-free cell culture medium that has a defined chemical compositionand supports proliferation of human lymphocytes. A list of serum-freeculture medium useful in the present invention would include, withoutlimitation, LONZA XVIVO-5, XVIVO-10, XVIVO-20, Sigma StemLine I,StemLine II, Yssel's media and AimV media.

Exemplary serum-free medium is described in the Example sectionpresented herein. An exemplary serum-free medium is serum-free XVIVO-20(Lonza), which may optionally be supplemented withpenicillin-streptomycin.

MEM and RPMI are protein-free basal media that do not contain growthfactors required for lymphocytes. A skilled practitioner wouldappreciate that a protein-free basal media can, however, be converted toserum-free media capable of supporting lymphocyte proliferationfollowing addition of required growth factors. Such serum-free mediacontain specific and defined growth factors (e.g., insulin) that arerequired for lymphocyte proliferation.

In Vitro Methods

As described herein, the present invention is directed to a method forpromoting differentiation and proliferation of human T helperlymphocytes that express IL17 (Th-IL17+ cells) in vitro, the methodcomprising the steps of: isolating a population of naive CD4+ T cellsfrom a human; and incubating the population of naive CD4+ T cells inserum-free culture medium comprising TGF-β, IL-1β, and any one of IL-6,IL-21 or IL-23, wherein the incubating promotes differentiation of humanTh-IL17+ cells.

As also described herein, the invention is directed to a method forgenerating a population of human T helper lymphocytes that express IL17(Th-IL17+ cells) in vitro, the method comprising the steps of: isolatinga population of naive CD4+ T cells from a human; and incubating thepopulation of naive CD4+ T cells in serum-free culture medium comprisingTGF-β, IL-1β, and any one of IL-6, IL-21 or IL-23, wherein theincubating promotes differentiation and proliferation of human Th-IL17+cells and thereby generates a population of human Th-IL17+ cells.

The in vitro methods of the invention are based on the novel andsurprising discoveries of the present inventors. In short, the presentinventors have devised an in vitro method for promoting human Th-IL17+cell differentiation and proliferation, which also provides a method forgenerating in vitro a population of human Th-IL17+ cells. The methodcalls for incubation of naive CD4+ T cells in serum-free mediasupplemented with TGF-β, IL-1β, and any one of IL-6, IL-21 or IL-23. Asdescribed herein, “serum-free media comprising TGF-β, IL-1β, and any oneof IL-6, IL-21 or IL-23”, refers to serum-free media comprising TGF-β,IL-1β, and any one of IL-6, IL-21 or IL-23 in amounts effective forpromoting human Th-IL17+ cell differentiation. Conditions wherein naiveCD4+ cells are incubated in serum-free media comprising TGF-β, IL-1β,and any one of IL-6, IL-21 or IL-23 may also be referred to herein ashuman Th-IL17+ cell promoting conditions. A negative control for humanTh-IL17+ cell promoting conditions is a matched serum-free media withoutcytokine supplementation (i.e., without TGF-β, IL-1β, and any one ofIL-6, IL-21 or IL-23). An exemplary negative control for human Th-IL17+cell promoting conditions is a matched serum-free media alone. It is tobe understood that supplementation with standard media additives forprevention of bacterial or fungal infection (such as, e.g.,penicillin-streptomycin) is not precluded from the method of the presentinvention. Indeed, in a particular embodiment of the present invention,the method calls for serum-free media comprising TGF-β, IL-1β, and anyone of IL-6, IL-21 or IL-23, which is supplemented withpenicillin-streptomycin. It is to be further understood that humanTh-IL17+ cell promoting conditions may also include additional cytokinesupplementation as described herein.

As taught herein, in vitro differentiation of human Th-IL17+ cells maybe evaluated or measured by detecting an increase in the expression of ahuman Th-IL17+ cell marker, such as IL17, IL17F, IL23R, RORC or IL26, ina population of CD4+ T cells incubated in accordance with the presentmethod. Each of these cellular molecules (IL17, IL17F, IL23R, RORC orIL26) serves as a positive marker indicative of human Th-IL17+ celldifferentiation. Indeed, expression of IL17, IL17F, IL23R, RORC or IL26is induced by 10- to about 100-fold in CD4+ cells incubated in humanTh-IL17+ cell promoting conditions relative to those treated undernegative control conditions. Accordingly, a change in the expression ofat least one of these markers reflects a differential in human Th-IL17+cell differentiation. More particularly, a change in the expression ofat least one of these markers reflects a differential in human Th-IL17+cell differentiation in a population of CD4+ cells incubated in humanTh-IL17+ cell promoting conditions. A change in expression of any ofthese markers may be determined using a variety of experimentalprotocols, including, but not limited to, real-time PCR usingappropriate primers. Experimental protocols that can be used todetermine expression of such markers and relative expression levels aredescribed in detail herein and are understood in the art.

In serum-free conditions in the absence of TGF-β, IL-1β, and any one ofIL-6, IL-21 or IL-23, Th17 markers do not increase. The presentinventors were, however, the first to define intermediate conditions,such as, e.g., that TGFβ alone induces RORγT expression and CCR6expression, but not IL-17. Such intermediate conditions represent thoseconditions necessary to achieve a partial progression along the pathwayof Th-17+ cell differentiation.

Agents

As used herein, an “agent”, “candidate compound”, or “test compound” maybe used to refer to, for example, nucleic acids (e.g., DNA and RNA),carbohydrates, lipids, proteins, peptides, peptidomimetics, smallmolecules and other drugs. More particularly an agent may refer toazole-containing compounds, cholesterol derivative compounds, retinoidderivative compounds, short hairpin RNA (shRNA), small interfering RNA(siRNA), neutralizing and/or blocking antibodies, tryptophan derivativecompounds, Vitamin D derivatives, or molecules known to inhibit fever,inflammation, or regulatory T (Treg) cell differentiation.

A short hairpin RNA (shRNA) is a sequence of RNA that makes a tighthairpin turn that can be used to silence gene expression via RNAinterference. shRNA is generally expressed using a vector introducedinto cells, wherein the vector utilizes the U6 promoter to ensure thatthe shRNA is always expressed. This vector is usually passed on todaughter cells, allowing the gene silencing to be inherited. The shRNAhairpin structure is cleaved by the cellular machinery into siRNA, whichis then bound to the RNA-induced silencing complex (RISC). This complexbinds to and cleaves mRNAs that match the siRNA to which it is bound.

Small interfering RNA (siRNA), sometimes known as short interfering RNAor silencing RNA, are a class of 20-25 nucleotide-long double-strandedRNA molecules that play a variety of roles in biology. Most notably,siRNA is involved in the RNA interference (RNAi) pathway whereby thesiRNA interferes with the expression of a specific gene.

Azole-containing compounds are chemical compounds that contain at leastan azole group. Azole groups are five-membered nitrogen heterocyclicring compounds containing at least one other noncarbon atom, nitrogen,sulfur or oxygen. Azole groups are aromatic and have two double bonds.Azole-containing compounds comprise the family of Imidazole and triazoleantifungals (e.g. Clotrimazole).

Sterol derivative compounds are compounds that are, for instance,intermediate in the pathway of cholesterol synthesis (mevalonatepathway). That includes lanosterol, FF-MAS (follicularfluid-meiosis-activating sterol).

Retinoid derivatives are compounds structurally related to Vitamin A andinclude, without limitation, retinoic acid.

Tryptophan derivative compounds are compounds that are generated fromthe degradation of tryptophan, through the action of, for instance,metabolism (Action of the IDO enzyme) or UVB radiation.

Vitamin D derivatives are compounds that are structurally related toVitamin D and that include 1-25VitaminD3.

As described herein, an agent identified using the method of the presentinvention that is a “modulator of human Th-IL17+ cell differentiation”is defined as an agent that is capable of modulating (e.g., increasingor decreasing) in vitro differentiation of human Th-IL17+ cells. Such anagent may be identified by its ability to effect a change in theexpression of a human Th-IL17+ cell marker, such as IL17, IL17F, IL23R,RORC or IL26, in a population of CD4+ T cells incubated in humanTh-IL17+ cell promoting conditions. As described herein, each of thesecellular molecules (IL17, IL17F, IL23R, RORC or IL26) serves as apositive marker indicative of human Th-IL17+ cell differentiation.Expression of IL17, IL17F, IL23R, RORC or IL26 can be induced by 10- to100-fold in CD4+ T cells treated to promote Th-IL17+ celldifferentiation relative to those treated under negative controlconditions in accordance with the method of the present invention. Incontrast, FOXP3 expression is inversely correlated with human Th-IL17+cell differentiation. FOXP3, therefore, serves as a negative marker ofhuman Th-IL17+ cell differentiation. Accordingly, a change in theexpression of at least one of these markers (positive or negative)responsive to the presence of an agent reflects a differential in humanTh-IL17+ cell differentiation. More particularly, a change in theexpression of at least one of these markers reflects a differential inhuman Th-IL17+ cell differentiation in a population of cells incubatedin human Th-IL17+ cell promoting conditions, wherein the change isdependent on incubation in the presence of a particular agent. Asdetailed below, experimental protocols of utility in determiningexpression of such markers and relative expression levels are describedin detail herein and are understood in the art. Such experimentalprotocols, include, but are not limited to, real-time PCR usingappropriate primers.

As taught herein, the change effected by an agent that is a modulator ofhuman Th-IL17+ cell differentiation is determined relative to that of apopulation of CD4+ T cells incubated in parallel in the absence of theagent or in the presence of a control agent (as described below), eitherof which is analogous to a negative control condition.

In accordance with the present invention, the method described hereinmay be used to achieve an increase in the number of human Th17+ cells ina cell population incubated in human Th-IL17+ cell promoting conditions,as described herein. An increase in the number of human Th17+ cells insuch a cell population may be expressed as the percent (%) of humanTh17+ cells present in such a cell population relative to the totalnumber of cells. In accordance with the present invention, the methoddescribed herein typically achieves 1% to 15% human Th17+ cells in acell population. It will be appreciated, however, that the presentmethod may be used to achieve a higher relative percent human Th17+cells in a cell population. Accordingly, the present invention is not inany way limited to achieving 1% to 15% human Th17+ cells in a treatedcell population.

In light of the above, it will be appreciated that an agent identifiedusing the method of the present invention that is a “modulator of humanTh-IL17+ cell differentiation” may be identified by its ability toeffect a change in the percent of human Th17+ cells in a population ofCD4+ T cells incubated in human Th-IL17+ cell promoting conditions. Astaught herein, a change in the percent of human Th17+ cells in apopulation of CD4+ T cells incubated in the presence of an agent isdetermined relative to the percent of human Th17+ cells in a populationof CD4+ T cells incubated in the absence of the agent or in the presenceof a control agent (negative control condition).

The term “control substance”, “control agent”, or “control compound” asused herein refers a molecule that is inert or has no activity relatingto an ability to modulate a biological activity. With respect to thepresent invention, such control substances are inert with respect to anability to modulate human Th-IL17+ cell differentiation in vitro.Exemplary controls include, but are not limited to, solutions comprisingphysiological salt concentrations.

In accordance with the present invention, incubation in the presence ofan agent that results in a decrease in expression of a human Th-IL17+cell marker, such as IL17, IL17F, IL23R, RORC or IL26, indicates thatthe agent is an inhibitor of human Th-IL17+ cell differentiation invitro. An agent that results in a decrease in expression of a humanTh-IL17+ cell marker, such as IL17, IL17F, IL23R, RORC or IL26, isreferred to herein as an inhibitor of human Th-IL17+ celldifferentiation. An inhibitor of human Th-IL17+ cell differentiation isan agent that effects at least a 2-fold decrease in the expression of ahuman Th-IL17+ cell marker, such as IL17, IL17F, IL23R, RORC or IL26.More particularly, an inhibitor of human Th-IL17+ cell differentiationis an agent that effects at least a 3-fold decrease in the expression ofa human Th-IL17+ cell marker, such as IL17, IL17F, IL23R, RORC or IL26.The above fold decreases may be determined relative to human Th-IL17+cell marker expression levels induced by incubation in human Th-IL17+cell promoting conditions in the absence of the agent.

In accordance with the present invention, incubation in the presence ofan agent that results in an increase in expression of a human Th-IL17+cell marker, such as IL17, IL17F, IL23R, RORC or IL26, indicates thatthe agent is a promoter of human Th-IL17+ cell differentiation in vitro.An agent that results in an increase in expression of a human Th-IL17+cell marker, such as IL17, IL17F, IL23R, RORC or IL26, is referred toherein as a promoter of human Th-IL17+ cell differentiation. A promoterof human Th-IL17+ cell differentiation is an agent that effects at leasta 2-fold increase in the expression of a human Th-IL17+ cell marker,such as IL17, IL17F, IL23R, RORC or IL26. More particularly, a promoterof human Th-IL17+ cell differentiation is an agent that effects at leasta 3-fold increase in the expression of a human Th-IL17+ cell marker,such as IL17, IL17F, IL23R, RORC or IL26. The above fold increases maybe determined relative to human Th-IL17+ cell marker expression levelsinduced by incubation in human Th-IL17+ cell promoting conditions in theabsence of the agent.

Also in accordance with the present invention, incubation in thepresence of an agent that results in a decrease in expression of FOXP3(expression of which is inversely correlated with human Th-IL17+ celldifferentiation) indicates that the agent is a promoter or inducer ofhuman Th-IL17+ cell differentiation in vitro. An agent that results in adecrease in expression of FOXP3 is, therefore, referred to herein as apromoter or inducer of human Th-IL17+ cell differentiation. A promoteror inducer of human Th-IL17+ cell differentiation is an agent thateffects at least a 2-fold decrease in the expression of FOXP3. Moreparticularly, a promoter or inducer of human Th-IL17+ celldifferentiation is an agent that effects at least a 3-fold decrease inthe expression of FOXP3. The above fold decreases are determinedrelative to FOXP3 expression levels observed under matched controlconditions, but in the absence of the agent.

Also in accordance with the present invention, incubation in thepresence of an agent that results in an increase in expression of FOXP3(expression of which is inversely correlated with human Th-IL17+ celldifferentiation) indicates that the agent is an inhibitor of humanTh-IL17+ cell differentiation in vitro. An agent that results in anincrease in expression of FOXP3 is, therefore, referred to herein as aninhibitor of human Th-IL17+ cell differentiation. An inhibitor of humanTh-IL17+ cell differentiation is an agent that effects at least a 2-foldincrease in the expression of FOXP3. More particularly, an inhibitor ofhuman Th-IL17+ cell differentiation is an agent that effects at least a3-fold increase in the expression of FOXP3. The above fold increases aredetermined relative to FOXP3 expression levels observed under matchedcontrol conditions, but in the absence of the agent.

In accordance with the present invention, incubation in the presence ofan agent that results in a decrease in the percent of human Th17+ cellsgenerated indicates that the agent inhibits human Th-IL17+ celldifferentiation in vitro. Such an agent is referred to herein as aninhibitor of human Th-IL17+ cell differentiation. An inhibitor of humanTh-IL17+ cell differentiation is an agent that effects at least a 2-folddecrease in the percent of human Th17+ cells generated. Moreparticularly, an inhibitor of human Th-IL17+ cell differentiation is anagent that effects at least a 3-fold decrease in the percent of humanTh17+ cells generated. The above fold decreases may be determinedrelative to the percent of human Th17+ cells generated by incubation inhuman Th-IL17+ cell promoting conditions in the absence of the agent.

In accordance with the present invention, incubation in the presence ofan agent that results in an increase in the percent of human Th17+ cellsgenerated indicates that the agent promotes human Th-IL17+ celldifferentiation in vitro. Such an agent is referred to herein as apromoter/inducer of human Th-IL17+ cell differentiation. A promoter orinducer of human Th-IL17+ cell differentiation is an agent that effectsat least a 2-fold increase in the percent of human Th17+ cellsgenerated. More particularly, a promoter/inducer of human Th-IL17+ celldifferentiation is an agent that effects at least a 3-fold increase inthe percent of human Th17+ cells generated. The above fold increases maybe determined relative to the percent of human Th17+ cells generated byincubation in human Th-IL17+ cell promoting conditions in the absence ofthe agent.

It is to be understood that agents capable of modulating human Th-IL17+cell differentiation, as determined using the in vitro method describedherein, are likely to exhibit similar modulatory capacity inapplications in vivo.

Modulatory agents identified using the screening methods of the presentinvention and compositions thereof can thus be administered fortherapeutic treatments. In therapeutic applications, modulatory agentsthat inhibit Th17+ T cell differentiation (i.e., inhibitors of Th17+ Tcell differentiation) and compositions thereof are administered to apatient suffering from an inflammatory or autoimmune disorder in anamount sufficient to at least partially arrest a symptom or symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective amount or dose.” Amountseffective for this use will depend on the severity of the disease andthe weight and general state of the patient.

Examples of inflammatory or autoimmune disorders that may be treatedusing inhibitors of Th17+ T cell differentiation include, withoutlimitation, multiple sclerosis, rheumatoid arthritis, Crohn's disease,and ulcerative colitis.

Methods for Determining Expression Levels of Human Th-IL17+ Cell Markers

Based on the guidance presented herein and knowledge in the relevantscientific fields, the expression level of a cellular marker of humanTh-IL17+ cells can be determined using a variety of techniques.Exemplary markers of human Th-IL17+ cell differentiation include, butare not limited to, IL17, IL17F, IL23R, RORC and IL26. Expression ofFOXP3, on the other hand, is negatively correlated with human Th-IL17+cell differentiation. FOXP3 is, therefore, a negative marker of humanTh-IL17+ cell differentiation. Expression levels of such markers (eithera positive or a negative marker) may be assessed with respect toexpressed nucleic acid corresponding to a cell marker (e.g., mRNA, totalRNA) or with respect to polypeptides encoded by same. A variety ofstandard protocols may be used to determine, for example, RNA level,including, but not limited to: polymerase chain amplification anddetection of amplified products therefrom, ribonuclease protection(RNase protection) assay, and Northern blot analysis. The principles andgeneral procedures of each of these methods are described in, forexample, Dvorak et al. (Biomed Papers 147:131, 2003), which isincorporated herein in its entirety. The principles and generalprocedures of each of these methods are, moreover, known in the art. Ina particular embodiment of the invention, real-time PCR is used todetect gene expression of human Th-IL17+ cell markers.

Real-Time PCR

As taught herein, detection of IL-17, IL17F, IL23R, RORC or IL26 geneexpression may be used as a means to assess human Th-IL17+differentiation. Detection of these markers of human Th-IL17+differentiation, therefore, provides positive indicators or readouts forthe present method for promoting differentiation and proliferation ofhuman Th-IL17+ differentiation. The induction of these genes in Th17promoting conditions is at least 10-fold, and can achieve about 100-foldrelative to the levels of these genes in non-promoting conditions.Particulars relating to real-time PCR analysis are presented in theExamples, as are primers for amplification of the above-indicatedTh-IL17 markers. See Table 2.

In non-Th17 promoting conditions, there are detectable levels of theTh-IL17 marker genes at the level of RNA, but there is no detectableprotein. The absence of detectable protein, therefore, presents arelevant baseline against which to compare Th-IL17 marker proteinlevels.

Detection of FOXP3 gene expression may also be used as a means to assesshuman Th-IL17+ differentiation. Detection of this negative marker ofhuman Th-IL17+ differentiation provides a negative indicator or readoutfor the present method for promoting differentiation and proliferationof human Th-IL17+ differentiation. A reduction in FOXP3 gene expression,however, provides a positive indicator or readout for the present methodfor promoting differentiation and proliferation of human Th-IL17+differentiation. The reduction of FOXP3 expression in Th17 promotingconditions is at least two-fold, and can achieve about ten-fold relativeto the levels of this gene in non-promoting conditions.

A variety of protocols are available for measuring and/or detectingexpression levels of polypeptides. Protocols for detecting polypeptideexpression, such as, for example, immunohistochemistry andimmunoblotting, are known in the art. These protocols are generallyapplicable to detecting IL17, IL17F, IL23R, RORC, IL26, or FOXP3polypeptides. Particular methods for detecting IL17, IL17F, IL23R, RORC,IL26, or FOXP3 polypeptides are described in the Examples presentedherein, as are reagents for performing such methods.

In general, immunoassays for polypeptides typically comprise contactinga sample, such as a population of cells (e.g., incubated in Th17promoting conditions or lysates thereof) in the presence of an antibodythat specifically or selectively binds to a polypeptide in question,e.g., a detectably labeled antibody capable of identifying, theparticular polypeptide (e.g., IL-17), and detecting the bound antibodyby any of a number of techniques well-known in the art (e.g., Westernblot, ELISA, FACS).

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support that is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled antibody thatselectively or specifically binds to the particular polypeptide (e.g., aTh17 cell marker). The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on a solid support may then be detected by conventional means.

More particularly, Th-IL17 marker protein levels can be assessed by cellsurface staining for CCR6 and IL23R; ELISA for IL-17, IL-17F, and IL-26;intracellular staining for IL17, IL17F, IL26, FOXP3 (negative marker),and RORC; and Western Blot for IL-17, IL-17F, IL23R, RORC, IL26, FOXP3(negative marker).

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Particular supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

An antibody can be detectably labeled by linking same to an enzyme andusing the labeled antibody in an enzyme immunoassay (EIA) (Voller, A.,“The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, DiagnosticHorizons 2:1, Microbiological Associates Quarterly Publication,Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol. 73:482; Maggio, E. (ed.),1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. etal., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzymethat is bound to the antibody reacts with an appropriate substrate,particularly a chromogenic substrate, in such a manner as to produce achemical moiety detectable, for example, by spectrophotometric,fluorimetric or by visual means. Enzymes which can be used to detectablylabel the antibody include, but are not limited to, malatedehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,catalase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect a polypeptide through theuse of a radioimmunoassay (MA). The radioactive isotope can be detectedby such means as the use of a gamma counter or a scintillation counteror by autoradiography.

An antibody may also be labeled with a fluorescent compound. When thefluorescently labeled antibody is exposed to light of the properwavelength, its presence can be detected due to fluorescence emission.Among the most commonly used fluorescent labeling compounds arefluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine.

An antibody can also be detectably labeled using fluorescence emittingmetals such as¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

An antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is determined by detecting the presence of luminescence thatarises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label an antibody.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin.

The basic molecular biology techniques used to practice the methods ofthe invention are well known in the art, and are described for examplein Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York; Ausubel et al., 1988, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York; and Ausubelet al., 2002, Short Protocols in Molecular Biology, John Wiley & Sons,New York).

Agents Identified by the Screening Methods of the Invention

The invention provides methods for identifying agents (e.g., candidatecompounds or test compounds) that modulate (inhibit or promote) humanTh-IL17+ differentiation. Agents that are capable of inhibiting humanTh-IL17+ differentiation, as identified by the screening method of theinvention, are useful as candidate anti-inflammatory or anti-autoimmunedisorder therapeutics.

A list of inflammatory or anti-autoimmune disorders that may be treatedusing an agent identified using a method of the invention includes,without limitation: arthritis, diabetes, multiple sclerosis, uveitis,rheumatoid arthritis, psoriasis, osteoporosis, asthma, bronchitis,allergic rhinitis, chronic obstructive pulmonary disease,atherosclerosis, H. pylori infections and ulcers resulting from suchinfection, graft versus host disease following bone marrowtransplantation, and inflammatory bowel diseases. Inflammatory boweldiseases treatable using agents identified by the present methodsinclude Crohn's disease, ulcerative colitis, sprue and food allergies.An inflammatory disease or condition may involve any organ or tissue inwhich the presence of T_(H)17 cells has been demonstrated and/orimplicated in disease etiology. Other diseases known to produceimmunopathological damage in the host, which may benefit from treatmentwith an agent identified using a method of the invention, may beselected from the group consisting of Hepatitis C virus, Influenza,SARS, and respiratory syncytial virus. The involvement of T_(H)17related genes autotoxin and maspin also suggests that prostate andbreast cancers may be treated using an agent identified using a methodof the invention. Evidence that the balance of T_(H)17 and Treg cells isspecifically altered in human immunodeficiency virus (HIV) infectionsalso suggests that immunodeficiencies and HIV infection may be treatedusing an agent identified using a method of the invention.

Examples of agents, candidate compounds or test compounds include, butare not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates,lipids, proteins, peptides, peptidomimetics, small molecules and otherdrugs. Agents can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam (1997) Anticancer Drug Des. 12:145; U.S. Pat. No.5,738,996; and U.S. Pat. No. 5,807,683, each of which is incorporatedherein in its entirety by reference).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233, each of which isincorporated herein in its entirety by reference.

Libraries of compounds may be presented, e.g., presented in solution(e.g., Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam(1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl.Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith (19900 Science249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990)Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310), each of which is incorporated herein in its entirety byreference.

Therapeutic Uses of Agents Identified

The invention provides for treatment of inflammatory and/or autoimmunedisorders by administration of a therapeutic agent identified using theabove-described methods. Such agents include, but are not limited toproteins, peptides, protein or peptide derivatives or analogs,antibodies, nucleic acids, and small molecules.

The invention provides methods for treating patients afflicted with aninflammatory and/or autoimmune disorder comprising administering to asubject an effective amount of a compound identified by the method ofthe invention. In a particular aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is particularly ananimal, including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is more particularly a mammal, and mostparticularly a human. In a specific embodiment, a non-human mammal isthe subject.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid are described above; additionalappropriate formulations and routes of administration are describedbelow.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu (1987)J. Biol. Chem. 262:4429-4432), and construction of a nucleic acid aspart of a retroviral or other vector. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally, e.g., by localinfusion during surgery, topical application, e.g., by injection, bymeans of a catheter, or by means of an implant, said implant being of aporous, non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

In another embodiment, the compound can be delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al.(1980) Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann.Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, e.g., an inflammatory site, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)). Other controlled release systems are discussed in the review byLanger (1990, Science 249:1527-1533).

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an agent anda pharmaceutically acceptable carrier. In a particular embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin, incorporated in its entiretyby reference herein. Such compositions will contain a therapeuticallyeffective amount of the compound, preferably in purified form, togetherwith a suitable amount of carrier so as to provide the form for properadministration to the subject. The formulation should suit the mode ofadministration.

In a particular embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment of an inflammatory or autoimmune disorder (e.g., Crohn'sdisease) can be determined by standard clinical techniques based on thepresent description. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachsubject's circumstances. However, suitable dosage ranges for intravenousadministration are generally about 20-500 micrograms of active compoundper kilogram body weight. Suitable dosage ranges for intranasaladministration are generally about 0.01 pg/kg body weight to 1 mg/kgbody weight. Suppositories generally contain active ingredient in therange of 0.5% to 10% by weight; oral formulations preferably contain 10%to 95% active ingredient. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Nucleic Acids

The invention provides methods of identifying agents capable ofmodulating human Th-IL17+ cell differentiation. Accordingly, theinvention encompasses administration of a nucleic acid encoding apeptide or protein capable of modulating human Th-IL17+ celldifferentiation, as well as antisense sequences or catalytic RNAscapable of interfering with human Th-IL17+ cell differentiation.

Any suitable methods for administering a nucleic acid sequence availablein the art can be used according to the present invention.

Methods for administering and expressing a nucleic acid sequence aregenerally known in the area of gene therapy. For general reviews of themethods of gene therapy, see Goldspiel et al. (1993) Clinical Pharmacy12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann.Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) Science260:926-932; and Morgan and Anderson (1993) Ann. Rev. Biochem.62:191-217; May (1993) TIBTECH 11(5): 155-215. Methods commonly known inthe art of recombinant DNA technology which can be used in the presentinvention are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler(1990) Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY.

In a particular aspect, the compound comprises a nucleic acid encoding apeptide or protein capable of modulating human Th-IL17+ celldifferentiation, such nucleic acid being part of an expression vectorthat expresses the peptide or protein in a suitable host. In particular,such a nucleic acid has a promoter operably linked to the coding region,said promoter being inducible or constitutive (and, optionally,tissue-specific). In another particular embodiment, a nucleic acidmolecule is used in which the coding sequences and any other desiredsequences are flanked by regions that promote homologous recombinationat a desired site in the genome, thus providing for intrachromosomalexpression of the nucleic acid (Koller and Smithies (1989) Proc. Natl.Acad. Sci. USA 86:8932-8935; Zijlstra et al. (1989) Nature 342:435-438).

Delivery of the nucleic acid into a subject may be direct, in which casethe subject is directly exposed to the nucleic acid or nucleicacid-carrying vector; this approach is known as in vivo gene therapy.Alternatively, delivery of the nucleic acid into the subject may beindirect, in which case cells are first transformed with the nucleicacid in vitro and then transplanted into the subject, known as “ex vivogene therapy”.

In another embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286); by direct injection of naked DNA;by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont); by coating with lipids, cell-surface receptors or transfectingagents; by encapsulation in liposomes, microparticles or microcapsules;by administering it in linkage to a peptide which is known to enter thenucleus; or by administering it in linkage to a ligand subject toreceptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432), which can be used to target cell typesspecifically expressing the receptors.

In another embodiment, a nucleic acid-ligand complex can be formed inwhich the ligand comprises a fusogenic viral peptide to disruptendosomes, allowing the nucleic acid to avoid lysosomal degradation. Inyet another embodiment, the nucleic acid can be targeted in vivo forcell specific uptake and expression, by targeting a specific receptor(see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992 (Wu etal.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316 datedNov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarkeet al.), WO 93/20221 dated Oct. 14, 1993 (Young)). Alternatively, thenucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression, by homologous recombination (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al.(1989) Nature 342:435-438).

In a further embodiment, a retroviral vector can be used (see Miller etal. (1993) Meth. Enzymol. 217:581-599). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. Thenucleic acid encoding a desired polypeptide to be used in gene therapyis cloned into the vector, which facilitates delivery of the gene into asubject. More detail about retroviral vectors can be found in Boesen etal. (1994) Biotherapy 6:291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al. (1994) J. Clin. Invest. 93:644-651; Kiem et al. (1994) Blood83:1467-1473; Salmons and Gunzberg (1993) Human Gene Therapy 4:129-141;and Grossman and Wilson (1993) Curr. Opin. in Genetics and Devel.3:110-114.

Adenoviruses may also be used effectively in gene therapy. Adenovirusesare especially attractive vehicles for delivering genes to respiratoryepithelia. Adenoviruses naturally infect respiratory epithelia wherethey cause a mild disease. Other targets for adenovirus-based deliverysystems are liver, the central nervous system, endothelial cells, andmuscle. Adenoviruses have the advantage of being capable of infectingnon-dividing cells. Kozarsky and Wilson (1993) Current Opinion inGenetics and Development 3:499-503 present a review of adenovirus-basedgene therapy. Bout et al. (1994) Human Gene Therapy 5:3-10 demonstratedthe use of adenovirus vectors to transfer genes to the respiratoryepithelia of rhesus monkeys. Other instances of the use of adenovirusesin gene therapy can be found in Rosenfeld et al. (1991) Science252:431-434; Rosenfeld et al. (1992) Cell 68:143-155; Mastrangeli et al.(1993) J. Clin. Invest. 91:225-234; PCT Publication WO94/12649; andWang, et al. (1995) Gene Therapy 2:775-783. Adeno-associated virus (AAV)has also been proposed for use in gene therapy (Walsh et al. (1993)Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146).

Another suitable approach to gene therapy involves transferring a geneto cells in tissue culture by such methods as electroporation,lipofection, calcium phosphate mediated transfection, or viralinfection. Usually, the method of transfer includes the transfer of aselectable marker to the cells. The cells are then placed underselection to isolate those cells that have taken up and are expressingthe transferred gene. Those cells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr (1993) Meth.Enzymol. 217:599-618; Cohen et al. (1993) Meth. Enzymol. 217:618-644;Cline (1985) Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. In a particular embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the subject; recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, the condition of the subject,etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to neuronal cells, glial cells (e.g., oligodendrocytes orastrocytes), epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood orfetal liver. In a particular embodiment, the cell used for gene therapyis autologous to the subject that is treated.

In another embodiment, the nucleic acid to be introduced for purposes ofgene therapy may comprise an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby adjusting the concentration of an appropriate inducer oftranscription.

Direct injection of a DNA coding for a peptide or protein capable ofmodulating human Th-IL17+ cell differentiation may also be performedaccording to, for example, the techniques described in U.S. Pat. No.5,589,466. These techniques involve the injection of “naked DNA”, i.e.,isolated DNA molecules in the absence of liposomes, cells, or any othermaterial besides a suitable carrier. The injection of DNA encoding aprotein and operably linked to a suitable promoter results in theproduction of the protein in cells near the site of injection.

Homogeneous Populations of Human Th-IL17+ Cells

The novel methods of the present invention facilitate the generation ofa homogeneous population of human Th-IL17+ cells comprising about or atleast 10⁹ homogeneous human Th-IL17+ cells, wherein the human Th-IL17+cells do not express cellular markers of other differentiated Th(non-Th-IL17+) cells. More specifically, the isolated homogeneouspopulation of human Th-IL17+ cells generated from naive CD4+ cells donot express cellular markers characteristic of Th1, Th2, or Treg cells.It is understood that cellular markers characteristic of Th1, Th2, orTreg cells would be present in any population of Th-IL17+ cellsgenerated from memory Th cells. Cellular markers characteristic of Th1cells include, for example, IFN-γ; cellular markers of Th2 cellsinclude, for example, IL4 or IL13; and cellular markers of Treg cellsinclude, for example, FoxP3. Accordingly, a homogeneous population ofhuman Th-IL17+ cells of the present invention does not include cellsthat express IFN-γ, IL4, IL13 or FoxP3.

The isolated population of about or at least 10⁹ human Th-IL17+ cellsgenerated from naive CD4+ cells as described herein is, therefore,homogeneous with respect to the expression of only those cellularmarkers characteristic of Th-IL17+ cells. Exemplary markers of humanTh-IL17 cells are IL-17, IL-17F, IL-26, and IL23R.

Prior to the present method, an isolated, homogeneous population ofabout or at least about 10⁹ human Th-IL17+ cells, wherein the humanTh-IL17+ cells do not express cellular markers of other differentiatedTh (non-Th-IL17+) cells, had not been generated.

It is noteworthy in this regard that Th-IL17+ cells generated frommemory T cells are not homogenous because they also contain Th1/Th2/Tregcells, as well as cells expressing various combinations of thecytokines, including Th1/Th17 combinations

As used herein and understood in the art, a human T helper type 1 (Th1)cell is a human cell of the CD4 T cell lineage (CD3+CD4+) that expressesat least one marker of differentiation into a Th1 cell, such as IFNγ.

As used herein and understood in the art, a human T helper type 2 (Th2)cell is a human cell of the CD4 T cell lineage (CD3+CD4+) that expressesat least one marker of differentiation into a Th2 cell, such as IL-4 orIL-13.

As used herein and understood in the art, a human T regulatory (Treg)cell is a human cell of the CD4 T cell lineage (CD3+CD4+) that expressesat least one marker of differentiation into a Treg cell, such as FoxP3.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

It is to be understood that this invention is not limited to particularassay methods, or test agents and experimental conditions described, assuch methods and agents may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only the appended claims.

EXAMPLE I

To avoid having antigen-experienced cells and serum-derived TGF-β in theTh17 differentiation cultures, the present inventors used human cordblood CD4⁺ T cells and serum-free medium. Under these conditions,induction of IL-17 and other Th17 gene products was observed only ifTGF-β was added to the culture medium. In contrast to requirements inmouse T cell cultures, human Th17 cell differentiation was not observedwhen IL-6 or IL-21 was combined with TGF-β. Instead, IL-1β and any oneof IL-6, IL-21 or IL-23 were required in combination with TGF-β forinduction of IL-17 expression. As in mouse, RORγT was induced by TGF-βand was required and sufficient for expression of Th17 cell products,suggesting that the basic mechanism of Th17 cell differentiation isevolutionarily conserved.

In accordance with the novel and surprising results presented herein,the present inventors have developed a new method for promotingdifferentiation and proliferation of human T helper lymphocytes thatexpress IL17 (Th-IL17+ cells) in vitro. The instant method is also wellsuited to screening methods whereby modulators (inhibitors orpromoters/inducers) of human Th-IL17+ cell differentiation areidentified. The present method is also useful for generating homogeneouspopulations of human Th-IL17+ cells.

Material and Methods

Cell Purification

Blood samples were obtained from the New York Blood Center. Mononuclearcells were prepared from buffy coats of healthy adult donors or fromcord blood on FicollPAQUE gradients. CD4⁺ T cells were isolated on anautoMACS Pro using Miltenyi bead depletion of CD14⁺ and CD25⁺ cellsfollowed by positive selection of CD4⁺ cells. Cord blood CD4⁺ T cellswere >97% pure and 100% CD45RA⁺ and were used as such for initialtransduction experiments. Adult CD4⁺ T cell subsets and naive cord bloodCD4⁺ T cells were further purified respectively asCD3⁺CD4⁺CD25⁻CD45RO^(−/+) and CD3⁺CD4⁺CD25⁻HLA-DR⁻CD45RA⁺ by cellsorting on a FACSAria.

Cell Culture and Lentiviral Transduction

Cells were cultivated in either RPMI1640 (Invitrogen) supplemented with10% fetal bovine serum (Hyclone), penicillin-streptomycin, 2 mMglutamine, 10 mM HEPES, 1 mM pyruvate and 0.1 mM non-essential aminoacids or serum-free XVIVO-20 (Lonza) supplemented withpenicillin-streptomycin in a 37° C. 5% CO₂ incubator. CD4⁺ T cells werestimulated by addition of anti-mouse IgG magnetic beads (Pierce)previously coated with purified anti-CD3 and anti-CD28 at finalconcentrations of 1 bead/cell and 1 μg/ml of each antibody. Fortransduction experiments, cells were seeded at a concentration of 10⁶cells/ml in 24 well plates with anti-CD3/CD28 coated beads, 10 μg/mlpolybrene and 10 U/ml IL-2 at day 0. Lentiviral supernatants were addedat an MOI ranging from 1 to 10. Cells were washed at day 1 and split asneeded in the presence of IL-2. For the shRNA experiment, puromycin wasadded at day 2 at 2 μg/ml. For polarization experiments, cells wereseeded at a concentration of 5×10⁵ to 10⁶ cells/ml in U-bottom 96 wellplates with anti-CD3/CD28 coated beads. IL-2 at 10 U/ml was either addedat day 0 or day 3. Media was replaced at day 3 and cells were split inthe presence of IL-2. For long-term experiments, cells were split asneeded. In some cases, 10 ng/ml IL-1β (eBioscience), 10 ng/ml IL-6(eBioscience), 10 ng/ml IL-21 (Cell Sciences), 10 ng/ml IL-23(eBioscience), various concentrations of TGF-β1 (PeproTech),neutralizing soluble IL-21R (R&D) and neutralizing antibodies againstIL-2, IL-4, IL-6, IFNγ or TGF-β (1 μg/ml except noted otherwise; seeTable 1 for details) were added at day 0 and maintained throughout theexperiment. Cells were harvested at day 6 for intracellular staining andreal-time PCR analysis except where specified otherwise.

TABLE 1 Antibodies used. Clone/Catalog Antigen number Company CCR6-FITCFAB196F R&D CCR6-biotin 11A9 BD CD3-Alexa750Cy7 UCHT1 eBioscienceCD4-PacBlue OKT4 eBioscience CD25-APC 555434 BD CD45RA-PE HI100eBioscience CD45RO-APC UCHL1 eBioscience IL-17-APC eBio64CAP17eBioscience IL-17-FITC eBio64DEC17 eBioscience IL-22-PE IC7621P R&DIFNγ-PECy7 45.B3 eBioscience HLA-DR-FITC 555558 BD HSA-PE (mCD24) 553262eBioscience CD3 purified UCHT1 eBioscience CD28 purified CD28.2eBioscience TGF-β purified 1D11 R&D IL-2 purified 5334.21 R&D IL-4purified MP425D2 eBioscience IL-6 purified MQ2-13A5 eBioscience IFNγpurified NIB42 eBioscienceSurface and Intracellular Staining

For intracellular cytokine staining, cells were incubated for 5 hourswith 50 ng/ml PMA (Sigma), 500 ng/ml Ionomycin (Sigma), and GolgiStop(BD). Surface staining was performed by incubation with thecorresponding fluorescently labeled antibodies for 15 min on ice.Intracellular staining was performed using the Cytofix/Cytoperm bufferset (BD). Briefly, cells were fixed and permeabilized for 30 min at roomtemperature and stained in permeabilization buffer for 30 min at roomtemperature. Flow cytometric measures were performed on a LSR II (BDBiosciences) instrument and analyzed using FlowJo software (Tree StarInc.). Antibodies are detailed in Table 1. FOXP3 was stained using FOXP3staining buffers (eBioscience). The present inventors used the FJK-16santibody, which was originally reported as an anti-mouse/rat FoxP3antibody. Indeed, the present inventors have observed that, unlike mostanti-human FOXP3 antibodies available, this antibody robustly stainsendogenous and over-expressed human full-length FOXP3.

Plasmids and Lentiviral Production

Human RORγT was cloned from human thymus. A double FLAG tag (DFTC) wasadded at the N-terminus. Human RORα isoform d was cloned from peripheralCD4⁺ T cells. Human RORβ was cloned from U937 cells. cDNAs were clonedin an HIV-derived vector HDV-IRES-HSA or HDV-IRES-GFP⁶⁵. Human GATA-3and T-bet lentiviral vectors have been previously described⁵⁷. shRNAvectors were obtained from OpenBiosystem. shRNA-1 is TRCN33657 (targetsequence TCTGCAAGACTCATCGCCAAA; SEQ ID NO: 1) and shRNA-2 is TRCN33658(target sequence CGAGGATGAGATTGCCCTCTA; SEQ ID NO: 2), and pLKO.1purowas used as control. Viral supernatants were produced by transienttransfection of 293T cells with vector DNA, a VSV-G expression plasmidand the pCMVΔR8.9 GagPol expression vectors for the shRNA vectors. Viralparticles were concentrated by ultracentrifugation at 25,000 rpm for 2 hat 4° C., resuspended in PBS containing 1% BSA, aliquoted and frozen.

Real-Time PCR

RNA was extracted by TRIZOL (Invitrogen) and cDNA was synthesized withSuperscript II (Invitrogen) and random primers. cDNA was analyzed byreal-time quantitative PCR in triplicates by using iQ CYBR GreenSupermix (Bio-Rad) or QuantiTect Multiplex PCR mix (Qiagen) in theiCycler Sequence Detection System (Bio-Rad). The starting quantity (SQ)of the initial cDNA sample was calculated from primer-specific standardcurves by using the iCycler Data Analysis Software. The expression levelof each gene was normalized to beta-actin expression level using thestandard curve method. Fold changes were calculated by normalizing tothe first sample of each set. Error bars were calculated based ontriplicate measurements of each gene. The primer sets for real-time PCRare detailed in Table 2.

TABLE 2 Primers used. Orienta- Gene tion Sequence ACTB FGGACTTCGAGCAAGAGATGG; SEQ ID NO: 3 ACTB RAGCACTGTGTTGGCGTACAG; SEQ ID NO: 4 ACTB ProbeCTCTTCCAGCCTTCCTTCCT; SEQ ID NO: 5 RORA F CGGTGCCTTTGACTCTCAGAACAACACCG;SEQ ID NO: 6 RORA R TCTTTCCAAATTCAAACACAAAGC; SEQ ID NO: 7 RORA ProbeTTGATGGGAAGTATGCCAGC; SEQ ID NO: 8 RORC FTTTTCCGAGGATGAGATTGC; SEQ ID NO: 9 RORC RCTTTCCACATGCTGGCTACA; SEQ ID NO: 10 RORC ProbeAAGACTCATCGCCAAAGCAT; SEQ ID NO: 11 IL23R FCATGACTTGCACCTGGAATG; SEQ ID NO: 12 IL23R RGCTTGGACCCAAACCAAGTA; SEQ ID NO: 13 IL23R Probe TGATTCATTACAAGGTGGCAA;SEQ ID NO: 14 IL17F F TGAAGCTTGACATTGGCATC; SEQ ID NO: 15 IL17F RTTCCTTGAGCATTGATGCAG; SEQ ID NO: 16 IL17F ProbeACCTCCCCCTGGAATTACAC; SEQ ID NO: 17 IL17 FACCAATCCCAAAAGGTCCTC; SEQ ID NO: 18 IL17 RGGGGACAGAGTTCATGTGGT; SEQ ID NO: 19 IL17 ProbeGCAATGAGGACCCTGAGAGA; SEQ ID NO: 20 IL26 FTGCAAGGCTGCAAGAAAATA; SEQ ID NO: 21 IL26 RCCAGTTCACTGATGGCTTTG; SEQ ID NO: 22 IL26 ProbeGGCAGAAATTGAGCCACTGT; SEQ ID NO: 23 IL6 FAAAGAGGCACTGGCAGAAAA; SEQ ID NO: 24 IL6 RTTTCACCAGGCAAGTCTCCT; SEQ ID NO: 25 IL21 FTTCTGCCAGCTCCAGAAGAT; SEQ ID NO: 26 IL21 RTTGTGGAAGGTGGTTTCCTC; SEQ ID NO: 27 IL21 ProbeTGGTCAGCTTTTTCCTGCTT; SEQ ID NO: 28ResultsRORγt-Dependent IL-17 Expression in Human Memory CD4⁺ T Cells

To evaluate the effect of IL-1β on IL-17 production, the presentinventors sorted naive CD45RO⁻CD25⁻CCR7⁺ and Th17-containing memoryCD45RO⁺CD25⁻CCR6⁺CD4⁺ T cells from adult peripheral blood and culturedthem in serum-containing media in the presence or absence of IL-1β.IL-1β induced a two-fold increase of IL-17 production in CCR6⁺ memorycells, but had no effect on CD45RO⁻ cells (FIG. 1a ). Similar resultswere observed with CD45RA⁺ sorted cells. With the goal of identifyingrequirements for Th17 cell differentiation in humans, the presentinventors initially wished to evaluate whether RORγT was necessary inpre-committed Th17 cells to maintain effector function. To ablate RORγTexpression, two shRNAs that demonstrated potent knock-down of RORγT bytransient transfection were utilized. Sorted CD45RO⁺CCR6⁺ memory CD4⁺ Tcells isolated from adult blood were transduced with the shRNA vectors.After 6 days, shRNA-1 and shRNA-2 reduced RORγT expression by 50% and90%, respectively (FIG. 1b ). Correspondingly, the present inventorsobserved on average 2-fold and 3.2-fold decreases in IL-17⁺ cells withshRNA-1 and shRNA-2, respectively (FIG. 1c ). The proportion of IFNγ⁺cells remained high in all samples. Thus, RORγT is required formaintenance of IL-17 expression in differentiated T cells.

RORγT Induces Expression of IL-17, IL-17F, IL-26 and CCR6 in Human NaiveCord Blood CD4⁺ T Cells.

The present inventors next asked whether overexpression of RORγT, thatwould bypass any requirement of its induction by cytokines, would besufficient to obtain IL-17 expression in naive CD4⁺ human T cells. CD4⁺T cells were isolated from human cord blood to ensure a naive phenotype,activated with anti-CD3 and anti-CD28, and transduced with control orhuman RORγT-encoding lentivirus. IL-17 expression was readily detected,peaking at 6 days in cells transduced with the RORγT vector (FIG. 1d ).The proportion of interferon-γ-expressing cells was substantiallyreduced by the expression of RORγT (FIG. 1d ). RORαd and RORβ, two otherROR-family members, also induced IL-17 expression when over-expressed inprimary human T cells (FIG. 6).

The present inventors did not detect IL-22 protein induction with RORγToverexpression. This was unexpected because IL-22 mRNA is stronglyup-regulated by Rorγt in murine CD4⁺ T cells (L. Zhou et al.,unpublished observations). This apparent discrepancy between mouse andhuman led us to investigate the IL22 gene of various species (FIG. 7a ).In human, the IL22 gene is located in the same locus as IFNG and IL26.In mouse, there is no IL26 gene, and Il22 is located in the same locusas Ifng and Iltifb, an Il22 duplication. IL-26, similarly to IL-22, isan IL-10 related cytokine and is found in memory CD4⁺ T cells expressingIL-17¹⁵. Quantitative PCR analysis of RORγT-expressing cells indicatedthat IL-26 is induced along with IL-17 and IL-17F by RORγT (FIG. 1e ).The IL26 gene was also found in the genome of pre-placental vertebrates,including zebrafish³⁵, but not in the genome of rat and mouse (FIG. 7b), suggesting that it was lost in a common ancestor for both rodents.

Human Th17 cells are exclusively found in the CD45RO⁺CCR6⁺ compartmentin adult blood. However, this compartment also contains IFNγ⁺IL-17⁻ andIFNγ⁺IL-17⁺ cells. In order to determine which transcription factorcould induce CCR6 expression in CD4⁺ T cells, the present inventorstransduced cord blood CD4⁺ T cells with vectors encoding RORγT or thetranscription factors involved in specification of the Th1 and Th2 celllineages, T-bet and GATA-3, respectively, as well as a control emptyvector. CCR6 was induced in RORγT-expressing cells, but not in cellstransduced with GATA-3 or T-bet, and it was not induced in trans incultures of RORγT expressing cells (FIG. 1f ). Expression of CCR2 andCCR4, also suggested to be Th17 cell markers^(4,36), was not altered byoverexpression of RORγT.

Antagonistic Effects of TGF-β on RORγT Function

The present inventors next sought to determine how cytokines known toaffect Th17 cells in mouse or human would affect IL-17 expressionfollowing RORγT overexpression, circumventing the potential effect ofthose cytokines on RORγT expression per se. Cord blood CD4⁺ T cells weretransduced with RORγT alone or in the presence of IL-1β, IL-6, or IL-21in combination with various concentrations of TGF-β (FIG. 2a ). Additionof IL-1β, IL-6 and IL-21 increased by about two-fold the proportion ofIL-17-producing cells obtained after forced expression of RORγT.However, TGF-β potently suppressed IL-17 production. Interestingly,addition of any one of IL-1β, IL-6 or IL-21 partially relieved thesuppression induced by TGF-β. The present inventors then investedwhether these cytokines could influence expression of endogenous RORγT.CD45RO⁻ naive, as well as subsets of memory CD4⁺ T cells, were sortedfrom adult peripheral blood based on CCR6 and CCR4 expression forcomparison of RORγT mRNA levels. RORγT expression was enriched in CCR6⁺cells. Various cytokines were screened for their ability to induce theexpression of RORC, the gene encoding for RORγT, in CD45RO⁻ adult naiveCD4⁺ T cells. Surprisingly, addition of TGF-β alone induceddose-dependent RORγT expression, but none of the other cytokines hadsuch an effect (FIGS. 2b and 2c ). However, treatment with TGF-β aloneor with IL-1β, IL-6 or IL-21, was insufficient to induce significantIL-17 expression as detected by intracellular staining under theseconditions.

The observation that cultures containing IL-1β, IL-6 and IL-21 hadincreased IL-17 expression following RORγT transduction (FIG. 2a )suggested that an endogenous source of TGF-β existed in our cultureconditions, and that addition of the other cytokines relieved its effectin a similar manner to their effect following addition of exogenousTGF-β. Indeed TGF-β is found in human and bovine serum^(37,38).Furthermore, serum TGF-β was found to be sufficient to induce FOXP3expression in naive human CD4⁺ T cells³⁹. The present inventors thusasked whether IL-17 expression was increased in serum-free conditions.Cord blood CD4⁺ T cells were transduced with RORγT in RPMI-10% FBS andserum-free media. A significant increase in IL-17 production wasobserved in serum-free media, which was most pronounced for donors thatexhibit the lowest expression of IL-17 following RORγT transduction inthe presence of serum (FIG. 2d ). Concurrently, FOXP3 expression wasinduced in RPMI-10% FBS, but not in serum-free media. Following additionof a neutralizing anti-TGF-β antibody in RPMI-10% FBS, FOXP3 inductionwas almost abolished, while IL-17 expression was increased, but not tothe extent observed in serum-free media. Higher concentration ofneutralizing antibody did not improve IL-17 expression (FIG. 8).

These observations indicated that TGF-β present in serum inhibits tosome extent IL-17 expression induced by RORγT and that otherunidentified compounds contained in fetal bovine serum possibly alsocounteract Th17 cell differentiation. These findings prompted thepresent inventors to evaluate whether TGF-β, IL-1β, IL-6 and IL-21 wouldinduce IL-17 production in serum-free media.

TGF-β, IL-1β and Either IL-6, IL-23 or IL-21 are Required for Human Th17Cell Polarization in Serum-Free Media

Naive cord blood CD4⁺ T cells were activated with anti-CD3/CD28 beads inserum-free media in the presence of anti-IL-4 and anti-IFNγ alone orwith various combinations of cytokines. These combinations includedincreasing concentrations of TGF-β with no added cytokine or with IL-1β,IL-6 or IL-21, with or without IL-23 (FIG. 3a ). After two weeks ofculture, IL-17 expression could be detected by intracellular stainingonly in cells cultivated with a combination of TGF-β, IL-1β and IL-23.Although IL-2 was found to inhibit IL-17 expression in mice⁴⁰, IL-1βrelieved this effect⁴¹. The effect of IL-2 and neutralizing anti-IL-2antibody was then tested in the above-indicated culture conditions. Inthe presence of TGF-β, IL-1β and IL-23, IL-17 expression was increasedat day 6 when IL-2 was included (FIG. 3b ). Addition of an anti-IL-2blocking antibody prevented cell proliferation and IL-17 expressioncould not be detected. Thus IL-2 appears to have a positive effect onIL-17 expression in human CD4⁺ T cell culture. In the mouse, IL-23,IL-21 and IL-6 share the ability to activate STAT3 if their cognatereceptors are expressed. Although IL-6 and IL-21 failed to induce IL-17in human cord blood T cells when combined with TGF-β alone, they wereactive, albeit not as strongly as IL-23, when both IL-1β and TGF-β wereincluded (FIG. 3c ). IL-21 is synthesized in response to IL-6 by Th17cells in the mouse and acts in an autocrine manner to induce Th17 celldifferentiation²⁵⁻²⁷. The present inventors subsequently evaluated ifIL-21 and/or IL-6 were required for human IL-17 induction, as they arein mouse. In human cells, IL-6 and IL-21 expression was not induced byIL-1β+IL-23+TGF-β (FIGS. 9a and 9b ). Furthermore, addition ofneutralizing anti-IL-6 antibody or soluble IL-21R had no effect on IL-17expression at levels that inhibited STAT3 phosphorylation (FIG. 9c ).Thus, in contrast to observations with naive mouse CD4⁺ T cells, IL-23can participate in induction of IL-17 in human T cells in the absence ofIL-6 and IL-21.

TGF-β, IL-1β and IL-23 Induce the Expression of IL-17, IL-17F, IL-26,IL-23R, CCR6 and RORγT and Inhibit the Expression of FOXP3

In light of the above findings, the present inventors adopted acombination of cytokines containing TGF-β, IL-1β, IL-23, and IL-2 forTh17 cell polarization of human cord blood naive CD4⁺ T cells. IL-17⁺cells were clearly detected as early as day 3 and increased up to day 6in culture (FIG. 3d ). Across multiple cord blood samples, theproportion of IL-17⁺ cells obtained under these conditions ranged from0.5% to 11% (FIG. 3e ). As in the mouse⁴², IL-17 induction was inhibitedby addition of retinoic acid (FIG. 10). The present inventors alsoevaluated IL-22 levels in Th17 cell differentiation cultures comprisingcord blood cells. A substantial proportion of naive cord blood CD4⁺ Tcells spontaneously expressed IL-22 protein after 6 days of culture(FIG. 3f ). As the concentration of TGF-β was increased, IL-22expression was progressively inhibited.

Since it was observed that RORγT induces expression of IL-26, thepresent inventors asked whether IL-26 could similarly be induced inhuman cord blood cells cultured under Th17 cell differentiationconditions. When naive CD4⁺ T cells were stimulated in the presence ofIL-2 in serum free medium, IL-17 mRNA expression detected by real-timePCR at day 6 was maximal following induction by the combination IL-1β,IL-23, and TGF-β, consistent with the intracellular staining (FIG. 4a ).IL-26 expression was also detected and levels of IL-26 mRNA increasedwith the dose of TGF-β (FIG. 4b ). Some expression of IL-17F was inducedby IL-1β alone and IL-23 alone had no effect (FIG. 4c ). However, in thepresence of IL-23 and IL-1β increasing concentrations of TGF-βsynergized to induce maximum IL-17F expression. RORC expression wasgradually induced with increasing concentrations of TGF-β and wasenhanced by further addition of both IL-1β+IL-23, but not eithercytokine alone. In the same conditions, RORA expression was slightlyinduced by TGF-β and there was no further effect upon addition of IL-1βand IL-23 (FIG. 11a ). This observation is in agreement with the slightenrichment of RORA mRNA levels in memory CCR6⁺ cells compared to CCR6⁻cells (FIG. 11b ). In mice, IL-23R is induced by IL-6 or IL-21 butinhibited by high concentrations of TGFβ⁴³. In human cells, IL-23Rexpression was induced to some extent by IL-23 alone, but not IL-1β,consistent with another report²⁸ (FIG. 4e ). However, IL-23R expressionreached maximal levels in the presence of IL-1β and IL-23 withincreasing concentrations of TGF-β. This suggests that in the presenceof TGF-β and IL-1β, IL-23 induces expression of its own receptor througha positive feedback loop, leading to maximum expression and induction ofRORγT, IL-17 and IL-17F. CCR6 cell surface expression was induced byTGF-β alone (FIG. 5a ). In conditions that induce IL-17 expression,IL-17 was detected only in CCR6+ cells (FIG. 5b ). FOXP3 expression wasinduced by an increasing concentration of TGF-β (FIG. 5c ). Addition ofIL-23, but not IL-1β, was able to suppress FOXP3 expression (FIG. 5c ).IL-6 and IL-21 were also able to inhibit FOXP3 expression. Thus, theregulation of FOXP3 expression during Th17 cell differentiation issimilar in mouse and humans.

Discussion

Based on recent studies employing both in vitro culture systems andgenetic approaches, it is now clear that TGF-β acts in concert with thepro-inflammatory cytokines IL-6, IL-21, and IL-23 to induce thedifferentiation of Th17 cells in mice²⁰. Phosphorylation of Stat3 uponengagement of the inflammatory cytokine receptors^(27,44) and inductionof Rorγt expression are essential for murine Th17 celldifferentiation²¹. The requirement for TGF-β in Th17 celldifferentiation was initially surprising, since it was known to act asan anti-inflammatory cytokine, at least in part through its inductionand maintenance of regulatory T cells²². The function of TGF-β may bedependent on context and thresholds, favoring Th17 cell differentiationat low concentrations in the presence of inflammatory cytokines and Tregcell differentiation at high concentrations⁴³.

In light of its pivotal role in controlling the Th17 vs. Treg balance inmice, it was unclear why TGF-β was found to be inhibitory in theinduction of IL-17 in human CD4⁺ T cells with a naive surfacephenotype^(15,29). As described herein, the present inventors have usedserum-free medium to reveal that TGF-β indeed has an essential role inthe differentiation of naive human CD4⁺ T cells towards the Th17 celllineage, similar to what has been observed in mice. In human T cells,TGF-β induced RORγT expression yet paradoxically inhibited itstranscriptional activity, thus preventing expression of IL-17. Acombination of IL-1β and any one of IL-6, IL-21 or IL-23 relieved thisinhibition and also contributed to RORγT expression, leading toinduction of IL-17. Thus, similarly to the mouse, TGF-β is required forIL-17 expression in human T cells and additional transcription factorsinduced by IL-1β and IL-6, IL-21 or IL-23 may be involved in inducingIL-17 expression.

The present inventors have demonstrated a requirement for IL-23 for invitro human Th17 cell differentiation, which contrasts with what hasbeen observed in the mouse, where IL-23 is required only in vivo¹⁸.However, with low concentrations of TGF-β in mouse T cell culture, apositive effect of IL-23 on production of IL-17 was found⁴³. Therefore,the discrepancy between the mouse and human systems with regards toIL-23 may be due to different culture conditions or differentsensitivities to TGF-β. The inflammatory cytokines IL-6, IL-21 and IL-23share signaling pathways by activating both STAT1 and STAT3⁴⁵⁻⁴⁹, whileIL-1β is thought to activate IRAK1/2 through recruitment ofMyd88^(50,51). Thus, STAT3 is likely to be a common denominator in theinduction of RORγT and IL-17 expression in both species²⁰. The IL-1pathway is important for the in vivo induction of Th17 cells in themouse, but it does not appear to be required for polarization in vitroin the presence of serum^(52,53). It remains to be determined whether anunrecognized requirement downstream of the IL-1 receptor is also neededduring murine Th17 cell differentiation in vitro.

Previous observations showing inhibitory activity of TGF-β in human Th17cell differentiation were probably confounded by the use of serum andnon-optimal naive cell purification^(28,29). Indeed, TGF-β has long beenrecognized to be a “switch” cytokine highly context- andconcentration-dependent⁵⁴. The present inventors revealed an essentialeffect of exogenous TGF-β in Th17 cell differentiation by usingserum-free media, suggesting that TGF-β present in serum might havemasked this effect. However, neutralization of TGF-β did not stronglyabolish the inhibitory effect of serum on RORγT-directed IL-17expression, and TGF-β was not able to completely inhibit IL-17expression following RORγT over-expression in serum-free conditions.This indicates that the presence of unidentified inhibitory factors inthe serum synergize with TGF-β to counteract Th17 cell differentiation.

In addition to IL-17, the present inventors have shown that IL-17F andIL-26 are induced by RORγT overexpression and cytokine polarization.IL-26 has been shown to target epithelial cells and has been suggestedto play a role in mucosal immunity⁵⁵, which is consistent with itsinduction in Th17 cells. Rearrangements of the Il22/Ifng locus seem tohave occurred in the mouse/rat lineage leading to a loss of Il26, butthis cytokine may have a major role in host defense and inflammation inhumans. Expression of IL-22 in human T cells was inhibited by TGF-β, inaccordance with observations in mouse systems¹⁴.

While all IL-17⁺ cells were CCR6⁺ following Th17 cell differentiation,CCR6 expression was induced by TGF-β. This is in agreement with theobservation that FOXP3⁺ cells can also express CCR6⁵⁶. As expected,FOXP3 expression was gradually induced by TGF-β alone in serum-freeconditions. IL-6, IL-21 and IL-23 alone were able to suppress FOXP3expression, similar to the mouse²⁰. However, addition of IL-1β wasrequired for the induction of IL-17 through a currently undefinedmechanism.

In human memory cells, expression of both IL-17 and IFNγ is clearlydetected, posing the question of their ontogeny. The polarizationconditions described here for naive cells lead only to the generation ofIL-17⁺IFNγ⁻ cells. It is likely that the high concentration of TGF-βrequired for Th17 polarization inhibits IFNγ expression. It remains tobe determined whether IL-17 and IFNγ can be expressed simultaneouslyfollowing T cell differentiation from naive cells. Functional plasticityin T helper cell differentiation has been observed⁵⁷. Thus, it ispossible that IL-17 or IFNγ is expressed only subsequently from a poolof differentiated Th1 or Th17 memory cells, respectively.

The present inventors previously demonstrated that the orphan nuclearreceptor Rorγt is uniquely expressed in mouse T cells that produce IL-17and is required for up-regulation of this cytokine in T cells both invivo and in vitro²¹. The present inventors show herein that RORγT alsohas a central role in human Th17 cell differentiation. shRNA knockdownof RORγT in memory CCR6⁺ cells resulted in a marked reduction of IL-17expression, indicating that the nuclear receptor is required formaintenance of cytokine production in Th17 cells. This result does notrule out a small contribution in directing IL-17 expression by theclosely related paralogue RORα, which was shown to have a similar rolein mouse⁵⁸. A strong enrichment of RORα mRNA in CCR6⁺ cells compared toCCR6⁻ cells was not, however, observed. Furthermore, RORα mRNA was notstrongly induced by a combination of IL-1β, IL-23 and TGFβ thatotherwise induced a 50-fold increase of RORγT mRNA. At least fourdifferentially spliced isoforms of RORα have, however, been describedand their respective transcriptional regulation has not been determined.Furthermore, the data herein do not exclude potentialpost-transcriptional regulation of RORα. Finally, the ability of RORβ toinduce IL-17 expression needs to be evaluated in relevant cell types,since RORβ expression has not been detected in peripheral CD4⁺ T cells.

The IL-23/Th17 axis has recently been implicated in multiple humandiseases⁵⁹. The present demonstration of a requirement for IL-23 in thedifferentiation of Th17 cells is relevant in light of multiplepolymorphisms in the human IL23R gene that have been reported to beassociated with Crohn's disease and psoriasis^(19,60-64). It will beimportant to elucidate the roles of IL-1β, IL-6, IL-21 and IL-23 andTGF-β in the pathogenesis of human diseases involving Th17 cells.Importantly, the participation of TGF-β in the induction of Th17 andTreg cells will likely be critical in maintenance of immune systemhomeostasis, particularly at mucosal surfaces, and imbalance in thissystem may result in autoimmunity. In this context, the present resultsoffer a working model for the study of human Th17 cell differentiationand provide new opportunities for manipulating these cells ininflammatory diseases.

EXAMPLE II

Experimental Methods

Mononuclear cells were prepared from human cord blood on FicollPAQUEgradients. CD4⁺ T cells were isolated on an autoMACS Pro using Miltenyibead depletion of CD14⁺ and CD25⁺ cells followed by positive selectionof CD4⁺ cells. Cord blood CD4⁺ T cells were >97% pure and 100% CD45RA⁺and were used as such for initial transduction experiments. Naive cordblood CD4⁺ T cells were further purified respectively asCD3⁺CD4⁺CD25⁻HLA-DR⁻CD45RA⁺ by cell sorting on a FACSAria.

CD4⁺ T cells were stimulated by addition of anti-mouse IgG magneticbeads (Pierce) previously coated with purified anti-CD3 and anti-CD28 atfinal concentrations of 1 bead/cell and 1 pg/ml of each antibody. Cellswere seeded at a concentration of 5×10⁵ to 10⁶ cells/ml in U-bottom 96well plates with anti-CD3/CD28 coated beads. IL-2 at 10 U/ml was addedat day 0. For Th17 differentiation, 10 ng/ml IL-1β (eBioscience), 10ng/ml IL-23 (eBioscience), and 10 ng/ml TGF-β1 (PeproTech) were added atday 0 and maintained throughout the experiment.

Cells were harvested at day 6. Total RNA was isolated using Trizolfollowing the manufacturer's procedure. RNA was labeled using thestandard 1-cycle procedure from Affymetrix. RNA was hybridized andanalyzed on Affymetrix HGU133plus2 chips. Data was analyzed using the Rpackage.

Results

The present inventors generated the expression profile of human cordblood naive CD4+ T cells cultured for 6 days in IL-2 (neutralconditions) or IL-2+IL-1β+IL-23+TGFβ (Th17 conditions). The relativeexpression fold change in the expression of each gene was determined bycomparing the expression level of each gene in Th17 conditions versusneutral conditions. The present inventors have thus identified a list ofgenes that are either up-regulated or down-regulated by at least 2 fold.See Tables 3 and 4.

In accordance with the present invention, therefore, novel cellularmarkers characteristic of human Th-IL17+ cells are identified. Suchcellular markers may contribute to functional properties of humanTh-IL17+ cells. Accordingly, the identification of such cellular markersof human Th-IL17+ cells provides novel targets for therapeutic agentsand intervention designed to modulate human Th-IL17+ celldifferentiation and/or activity.

TABLE 3 Human genes that are up-regulated in Th17 conditions. Genesknown to be up-regulated in the Th17 pathway are highlighted in bold asan indication of the validity of the data. fold order gene name changedescription 1 IL17A 460.3 interleukin 17A 2 SOX2 64.1 SRY (sexdetermining region Y)-box 2 3 IL17F 50.0 interleukin 17F 4 CXCL13 49.3chemokine (C—X—C motif) ligand 13 (B-cell chemoattractant) 5 DIXDC1 36.8DIX domain containing 1 6 DHRS9 30.0 dehydrogenase/reductase (SDRfamily) member 9 7 PTHLH 26.6 parathyroid hormone-like hormone 8 COL15A124.0 collagen, type XV, alpha 1 9 DSE 22.5 dermatan sulfate epimerase 10SERPINA1 22.4 serpin peptidase inhibitor, clade A (alpha- 1antiproteinase, antitrypsin), member 1 11 IL23R 22.2 interleukin 23receptor 12 FNBP1L 21.6 formin binding protein 1-like 13 IL9 21.4interleukin 9 14 KISS1R 16.7 KISS1 receptor 15 GATM 16.5 glycineamidinotransferase (L- arginine:glycine amidinotransferase) 16 BASP116.2 brain abundant, membrane attached signal protein 1 17 THBS1 15.9thrombospondin 1 18 SH3RF1 13.6 SH3 domain containing ring finger 1 19CCNA1 12.2 cyclin A1 20 CXCR4 12.0 chemokine (C—X—C motif) receptor 4 21IL1A 11.9 interleukin 1, alpha 22 CLIC5 11.2 chloride intracellularchannel 5 23 AQP3 11.1 aquaporin 3 (Gill blood group) 24 NAPSB 11.0napsin B aspartic peptidase pseudogene 25 HOP 10.4 homeodomain-onlyprotein 26 CCL20 10.3 chemokine (C-C motif) ligand 20 27 PLOD2 9.9procollagen-lysine, 2-oxoglutarate 5- dioxygenase 2 28 PTCHD1 9.9patched domain containing 1 29 CCR6 9.8 chemokine (C-C motif) receptor 630 IL1R2 9.5 interleukin 1 receptor, type II 31 RCAN2 9.5 regulator ofcalcineurin 2 32 BCAR3 9.2 breast cancer anti-estrogen resistance 3 33TIMP1 9.2 TIMP metallopeptidase inhibitor 1 34 EPAS1 9.2 endothelial PASdomain protein 1 35 MPP7 9.1 membrane protein, palmitoylated 7 (MAGUKp55 subfamily member 7) 36 KIF5C 8.7 kinesin family member 5C 37 PLEKHA58.7 pleckstrin homology domain containing, family A member 5 38 LTA 8.6lymphotoxin alpha (TNF superfamily, member 1) 39 RORA 8.5 RAR-relatedorphan receptor A 40 RGS16 8.4 regulator of G-protein signaling 16 41C13orf18 8.4 chromosome 13 open reading frame 18 42 TMEM154 8.3transmembrane protein 154 43 PRG4 8.3 proteoglycan 4 44 WBP5 8.3 WWdomain binding protein 5 45 DNM3 8.1 dynamin 3 46 LOC283666 7.9hypothetical protein LOC283666 47 KIAA0828 7.9 adenosylhomocysteinase 348 HBEGF 7.7 heparin-binding EGF-like growth factor 49 MRC1 7.5 mannosereceptor, C type 1 50 BMPR2 7.5 bone morphogenetic protein receptor,type II (serine/threonine kinase) 51 CTSL1 7.4 cathepsin L1 52 NA 7.1 NA53 EPHA4 6.9 EPH receptor A4 54 MYO10 6.8 myosin X 55 NRIP3 6.7 nuclearreceptor interacting protein 3 56 CXCL3 6.7 chemokine (C—X—C motif)ligand 3 57 LRP11 6.6 low density lipoprotein receptor-related protein11 58 PTK2 6.4 PTK2 protein tyrosine kinase 2 59 PERP 6.4 PERP, TP53apoptosis effector 60 SMARCA2 6.4 SWI/SNF related, matrix associated,actin dependent regulator of chromatin, subfamily a, member 2 61 DDIT46.4 DNA-damage-inducible transcript 4 62 SEPT10 6.4 septin 10 63 MGST16.2 microsomal glutathione S-transferase 1 64 RAB13 6.0 RAB13, memberRAS oncogene family 65 SLC1A1 6.0 solute carrier family 1(neuronal/epithelial high affinity glutamate transporter, system Xag),member 1 66 PALM2- 5.9 PALM2-AKAP2 protein AKAP2 67 UCHL1 5.9 ubiquitincarboxyl-terminal esterase L1 (ubiquitin thiolesterase) 68 RBMS3 5.7 RNAbinding motif, single stranded interacting protein 69 CRISPLD1 5.7cysteine-rich secretory protein LCCL domain containing 1 70 COL5A2 5.6collagen, type V, alpha 2 71 PGBD1 5.6 piggyBac transposable elementderived 1 72 CCDC88A 5.5 coiled-coil domain containing 88A 73 DBN1 5.4drebrin 1 74 RUNX1 5.4 runt-related transcription factor 1 (acutemyeloid leukemia 1; aml1 oncogene) 75 B4GALT6 5.4 UDP-Gal:betaGlcNAcbeta 1,4- galactosyltransferase, polypeptide 6 76 MAP1B 5.4microtubule-associated protein 1B 77 SNX7 5.4 sorting nexin 7 78 OSM 5.3oncostatin M 79 S100A2 5.3 S100 calcium binding protein A2 80 AYTL1 5.3acyltransferase like 1 81 LGALS3 5.3 lectin, galactoside-binding,soluble, 3 82 IL26 5.3 interleukin 26 83 COL6A3 5.2 collagen, type VI,alpha 3 84 ARMCX2 5.1 armadillo repeat containing, X-linked 2 85 DHRS25.1 dehydrogenase/reductase (SDR family) member 2 86 MLLT11 5.0myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog,Drosophila); translocated to, 11 87 CHN1 5.0 chimerin (chimaerin) 1 88ANXA3 5.0 annexin A3 89 GNA11 5.0 guanine nucleotide binding protein (Gprotein), alpha 11 (Gq class) 90 GALNT10 5.0 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 10(GalNAc-T10) 91 UPK1B 4.9 uroplakin 1B 92 RHPN2 4.9 rhophilin, RhoGTPase binding protein 2 93 DNAJC12 4.9 DnaJ (Hsp40) homolog, subfamilyC, member 12 94 PKIG 4.9 protein kinase (cAMP-dependent, catalytic)inhibitor gamma 95 C1orf218 4.9 chromosome 1 open reading frame 218 96PALLD 4.9 palladin, cytoskeletal associated protein 97 APOD 4.9apolipoprotein D 98 TPD52 4.8 tumor protein D52 99 PSCD3 4.8 pleckstrinhomology, Sec7 and coiled-coil domains 3 100 LOC286052 4.8 hypotheticalprotein LOC286052 101 HSDL2 4.7 hydroxysteroid dehydrogenase like 2 102SLC44A1 4.7 solute carrier family 44, member 1 103 FAM119A 4.7 familywith sequence similarity 119, member A 104 PNOC 4.7 prepronociceptin 105MOBKL2B 4.7 MOB1, Mps One Binder kinase activator- like 2B (yeast) 106ELK3 4.7 ELK3, ETS-domain protein (SRF accessory protein 2) 107 TRERF14.6 transcriptional regulating factor 1 108 PDE4DIP 4.6phosphodiesterase 4D interacting protein (myomegalin) 109 C10orf58 4.6chromosome 10 open reading frame 58 110 ELL2 4.6 elongation factor, RNApolymerase II, 2 111 LOC286144 4.5 hypothetical protein LOC286144 112CYB5R2 4.5 cytochrome b5 reductase 2 113 RORC 4.5 RAR-related orphanreceptor C 114 LOC285957 4.3 hypothetical protein LOC285957 115 PWCR14.3 Prader-Willi syndrome chromosome region 1 116 IL2RB 4.3 interleukin2 receptor, beta 117 OLAH 4.3 oleoyl-ACP hydrolase 118 RAB34 4.2 RAB34,member RAS oncogene family 119 SYT11 4.2 synaptotagmin XI 120 ABCG2 4.1ATP-binding cassette, sub-family G (WHITE), member 2 121 TMEM65 4.1transmembrane protein 65 122 RYR1 4.1 ryanodine receptor 1 (skeletal)123 PLEKHC1 4.0 pleckstrin homology domain containing, family C (withFERM domain) member 1 124 CCDC3 4.0 coiled-coil domain containing 3 125IMPA2 4.0 inositol(myo)-1(or 4)-monophosphatase 2 126 MMP2 4.0 matrixmetallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IVcollagenase) 127 C12orf39 4.0 chromosome 12 open reading frame 39 128CTSH 4.0 cathepsin H 129 NEFH 4.0 neurofilament, heavy polypeptide 200kDa 130 RAB23 4.0 RAB23, member RAS oncogene family 131 RTKN 4.0rhotekin 132 PLAGL1 4.0 pleiomorphic adenoma gene-like 1 133 SCARB2 3.9scavenger receptor class B, member 2 134 C14orf145 3.9 chromosome 14open reading frame 145 135 MST150 3.9 MSTP150 136 LIPG 3.9 lipase,endothelial 137 IDS 3.9 iduronate 2-sulfatase (Hunter syndrome) 138PITPNC1 3.8 phosphatidylinositol transfer protein, cytoplasmic 1 139FAM102B 3.8 family with sequence similarity 102, member B 140 HLX 3.8H2.0-like homeobox 141 CXCL12 3.8 chemokine (C—X—C motif) ligand 12(stromal cell-derived factor 1) 142 C9orf72 3.8 chromosome 9 openreading frame 72 143 TTC28 3.8 tetratricopeptide repeat domain 28 144DYNC2LI1 3.8 dynein, cytoplasmic 2, light intermediate chain 1 145 PHYH3.8 phytanoyl-CoA 2-hydroxylase 146 DMXL2 3.8 Dmx-like 2 147 TRPC1 3.8transient receptor potential cation channel, subfamily C, member 1 148C18orf1 3.8 chromosome 18 open reading frame 1 149 HSPA1A 3.8 heat shock70 kDa protein 1A 150 BNIP3L 3.8 BCL2/adenovirus E1B 19 kDa interactingprotein 3-like 151 ITGA2 3.7 integrin, alpha 2 (CD49B, alpha 2 subunitof VLA-2 receptor) 152 CLGN 3.7 calmegin 153 PIGF 3.7phosphatidylinositol glycan anchor biosynthesis, class F 154 INADL 3.7InaD-like (Drosophila) 155 PLS1 3.7 plastin 1 (I isoform) 156 GYS2 3.7glycogen synthase 2 (liver) 157 NGFRAP1 3.7 nerve growth factor receptor(TNFRSF16) associated protein 1 158 MYB 3.7 v-myb myeloblastosis viraloncogene homolog (avian) 159 TNFRSF12A 3.7 tumor necrosis factorreceptor superfamily, member 12A 160 RP13-36C9.3 3.7 cancer/testisantigen CT45-3 161 PLTP 3.7 phospholipid transfer protein 162 MIPEP 3.7mitochondrial intermediate peptidase 163 NPW 3.7 neuropeptide W 164 LTB3.6 lymphotoxin beta (TNF superfamily, member 3) 165 SORL1 3.6sortilin-related receptor, L(DLR class) A repeats-containing 166 ZNRF13.6 zinc and ring finger 1 167 TMEM5 3.6 transmembrane protein 5 168DFNA5 3.6 deafness, autosomal dominant 5 169 ITGAE 3.6 integrin, alpha E(antigen CD103, human mucosal lymphocyte antigen 1; alpha polypeptide)170 TCEAL4 3.6 transcription elongation factor A (SII)- like 4 171 CCR43.6 chemokine (C-C motif) receptor 4 172 AKAP12 3.6 A kinase (PRKA)anchor protein (gravin) 12 173 SWAP70 3.6 SWAP-70 protein 174 NTRK2 3.5neurotrophic tyrosine kinase, receptor, type 2 175 GPR160 3.5 Gprotein-coupled receptor 160 176 TPK1 3.5 thiamin pyrophosphokinase 1177 Rgr 3.5 Ral-GDS related protein Rgr 178 CSRP2 3.5 cysteine andglycine-rich protein 2 179 ALDH5A1 3.5 aldehyde dehydrogenase 5 family,member A1 (succinate-semialdehyde dehydrogenase) 180 TMEPAI 3.5transmembrane, prostate androgen induced RNA 181 PRKCDBP 3.5 proteinkinase C, delta binding protein 182 LIF 3.5 leukemia inhibitory factor(cholinergic differentiation factor) 183 IFT74 3.5 intraflagellartransport 74 homolog (Chlamydomonas) 184 LPGAT1 3.5lysophosphatidylglycerol acyltransferase 1 185 TARSL2 3.5 threonyl-tRNAsynthetase-like 2 186 NPAS2 3.5 neuronal PAS domain protein 2 187 TANC13.5 tetratricopeptide repeat, ankyrin repeat and coiled-coil containing1 188 DHTKD1 3.4 dehydrogenase E1 and transketolase domain containing 1189 TCFL5 3.4 transcription factor-like 5 (basic helix- loop-helix) 190GLB1 3.4 galactosidase, beta 1 191 HLA-DPB1 3.4 major histocompatibilitycomplex, class II, DP beta 1 192 RAB33A 3.4 RAB33A, member RAS oncogenefamily 193 ASPH 3.4 aspartate beta-hydroxylase 194 LOC401397 3.4hypothetical LOC401397 195 ACN9 3.4 ACN9 homolog (S. cerevisiae) 196B4GALT4 3.4 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase,polypeptide 4 197 hCG_1815491 3.4 hCG1815491 198 KIAA0888 3.4 KIAA0888protein 199 C3orf28 3.4 chromosome 3 open reading frame 28 200 RPP30 3.4ribonuclease P/MRP 30 kDa subunit 201 PDE6D 3.4 phosphodiesterase 6D,cGMP-specific, rod, delta 202 SYN1 3.4 synapsin I 203 MCAM 3.4 melanomacell adhesion molecule 204 UPP1 3.4 uridine phosphorylase 1 205 CASP63.4 caspase 6, apoptosis-related cysteine peptidase 206 LYN 3.4 v-yes-1Yamaguchi sarcoma viral related oncogene homolog 207 OCIAD2 3.4 OCIAdomain containing 2 208 LOC729680 3.4 hypothetical protein LOC729680 209KIAA1450 3.3 KIAA1450 protein 210 FRMD6 3.3 FERM domain containing 6 211SYNE1 3.3 spectrin repeat containing, nuclear envelope 1 212 PTGFRN 3.3prostaglandin F2 receptor negative regulator 213 TIFA 3.3TRAF-interacting protein with a forkhead-associated domain 214 TCEAL83.3 transcription elongation factor A (SII)- like 8 215 RLBP1L1 3.3retinaldehyde binding protein 1-like 1 216 HLA-DPA1 3.3 majorhistocompatibility complex, class II, DP alpha 1 217 NME4 3.3non-metastatic cells 4, protein expressed in 218 EPDR1 3.3 ependyminrelated protein 1 (zebrafish) 219 PNMA2 3.3 paraneoplastic antigen MA2220 MAN1C1 3.3 mannosidase, alpha, class 1C, member 1 221 CYFIP2 3.3cytoplasmic FMR1 interacting protein 2 222 LEPR 3.3 leptin receptor 223PSPH 3.3 phosphoserine phosphatase 224 HSPA1B 3.3 heat shock 70 kDaprotein 1B 225 EPOR 3.2 erythropoietin receptor 226 TRAF3IP1 3.2 TNFreceptor-associated factor 3 interacting protein 1 227 ENO2 3.2 enolase2 (gamma, neuronal) 228 GALM 3.2 galactose mutarotase (aldose 1-epimerase) 229 SOCS2 3.2 suppressor of cytokine signaling 2 230 CTLA43.2 cytotoxic T-lymphocyte-associated protein 4 231 ETV5 3.2 ets variantgene 5 (ets-related molecule) 232 SPRED1 3.2 sprouty-related, EVH1domain containing 1 233 THYN1 3.2 thymocyte nuclear protein 1 234 TM4SF13.2 transmembrane 4 L six family member 1 235 CBS 3.2cystathionine-beta-synthase 236 LPL 3.2 lipoprotein lipase 237 TGFBI 3.2transforming growth factor, beta-induced, 68 kDa 238 KL 3.2 klotho 239FAM92A1 3.2 family with sequence similarity 92, member A1 240 C22orf163.2 chromosome 22 open reading frame 16 241 TMEM110 3.2 transmembraneprotein 110 242 LRRC16 3.2 leucine rich repeat containing 16 243 TMEM1183.2 transmembrane protein 118 244 ADCY3 3.1 adenylate cyclase 3 245ATP8B2 3.1 ATPase, Class I, type 8B, member 2 246 TGFB1 3.1 transforminggrowth factor, beta 1 247 C11orf74 3.1 chromosome 11 open reading frame74 248 NT5DC1 3.1 5′-nucleotidase domain containing 1 249 SUHW2 3.1suppressor of hairy wing homolog 2 (Drosophila) 250 GRAMD3 3.1 GRAMdomain containing 3 251 PLA2G12A 3.1 phospholipase A2, group XIIA 252APAF1 3.1 apoptotic peptidase activating factor 1 253 GLT1D1 3.1glycosyltransferase 1 domain containing 1 254 DEPDC1 3.1 DEP domaincontaining 1 255 PVRIG 3.1 poliovirus receptor related immuno- globulindomain containing 256 TNRC8 3.1 trinucleotide repeat containing 8 257LPXN 3.1 leupaxin 258 LYPLAL1 3.1 lysophospholipase-like 1 259 AIF1 3.1allograft inflammatory factor 1 260 CORO2A 3.1 coronin, actin bindingprotein, 2A 261 TUSC3 3.1 tumor suppressor candidate 3 262 ADH5 3.1alcohol dehydrogenase 5 (class III), chi polypeptide 263 LOC647500 3.1similar to phosphodiesterase 4D interacting protein isoform 1 264 CCDC343.1 coiled-coil domain containing 34 265 SSX2IP 3.1 synovial sarcoma, Xbreakpoint 2 interacting protein 266 TBC1D9 3.1 TBC1 domain family,member 9 (with GRAM domain) 267 MORN2 3.1 MORN repeat containing 2 268LPHN1 3.0 latrophilin 1 269 C14orf143 3.0 chromosome 14 open readingframe 143 270 RLN2 3.0 relaxin 2 271 C6orf170 3.0 chromosome 6 openreading frame 170 272 ZYG11B 3.0 zyg-11 homolog B (C. elegans) 273ELOVL4 3.0 elongation of very long chain fatty acids (FEN1/Elo2,SUR4/Elo3, yeast)-like 4 274 TTC8 3.0 tetratricopeptide repeat domain 8275 ARMCX1 3.0 armadillo repeat containing, X-linked 1 276 TUBA1A 3.0tubulin, alpha 1a 277 TMEFF1 3.0 transmembrane protein with EGF-like andtwo follistatin-like domains 1 278 HLA-DMA 3.0 major histocompatibilitycomplex, class II, DM alpha 279 MYO1G 3.0 myosin IG 280 LOC157278 3.0hypothetical protein LOC157278 281 CCDC53 3.0 coiled-coil domaincontaining 53 282 C1orf25 3.0 chromosome 1 open reading frame 25 283STAMBPL1 3.0 STAM binding protein-like 1 284 ADRBK2 3.0 adrenergic,beta, receptor kinase 2 285 CDC42BPB 3.0 CDC42 binding protein kinasebeta (DMPK-like) 286 ZNF697 3.0 zinc finger protein 697 287 AIG1 3.0androgen-induced 1 288 S100A6 3.0 S100 calcium binding protein A6 289CKAP4 3.0 cytoskeleton-associated protein 4 290 RPL39L 2.9 ribosomalprotein L39-like 291 NLRP3 2.9 NLR family, pyrin domain containing 3 292TMEM14A 2.9 transmembrane protein 14A 293 MNDA 2.9 myeloid cell nucleardifferentiation antigen 294 RCAN3 2.9 RCAN family member 3 295 ZBTB8 2.9zinc finger and BTB domain containing 8 296 FTO 2.9 fat mass and obesityassociated 297 MIB1 2.9 mindbomb homolog 1 (Drosophila) 298 PPARG 2.9peroxisome proliferator-activated receptor gamma 299 ANLN 2.9 anillin,actin binding protein 300 SQRDL 2.9 sulfide quinone reductase-like(yeast) 301 AOF1 2.9 amine oxidase (flavin containing) domain 1 302LRRCC1 2.9 leucine rich repeat and coiled-coil domain containing 1 303SMYD3 2.9 SET and MYND domain containing 3 304 SCHIP1 2.9 schwannomininteracting protein 1 305 C2orf33 2.9 chromosome 2 open reading frame 33306 DMD 2.9 dystrophin (muscular dystrophy, Duchenne and Becker types)307 CCDC74B 2.9 coiled-coil domain containing 74B 308 PLCXD2 2.9phosphatidylinositol-specific phospholipase C, X domain containing 2 309LXN 2.9 latexin 310 LTB4DH 2.9 leukotriene B4 12-hydroxydehydrogenase311 TUBG1 2.9 tubulin, gamma 1 312 ECGF1 2.9 endothelial cell growthfactor 1 (platelet- derived) 313 SPIRE1 2.9 spire homolog 1 (Drosophila)314 HTATIP2 2.9 HIV-1 Tat interactive protein 2, 30 kDa 315 CRYZ 2.9crystallin, zeta (quinone reductase) 316 FAM33A 2.9 family with sequencesimilarity 33, member A 317 C3orf14 2.9 chromosome 3 open reading frame14 318 PLEKHA8 2.9 pleckstrin homology domain containing, family A(phosphoinositide binding specific) member 8 319 PPIL4 2.9peptidylprolyl isomerase (cyclophilin)- like 4 320 DOCK7 2.9 dedicatorof cytokinesis 7 321 SIAE 2.9 sialic acid acetylesterase 322 FKBP1B 2.9FK506 binding protein 1B, 12.6 kDa 323 MCM6 2.9 minichromosomemaintenance complex component 6 324 TMTC4 2.9 transmembrane andtetratricopeptide repeat containing 4 325 KLHDC5 2.9 kelch domaincontaining 5 326 DNAJC6 2.9 DnaJ (Hsp40) homolog, subfamily C, member 6327 PDLIM5 2.8 PDZ and LIM domain 5 328 PON2 2.8 paraoxonase 2 329FLJ13305 2.8 hypothetical protein FLJ13305 330 NELL2 2.8 NEL-like 2(chicken) 331 C18orf50 2.8 chromosome 18 open reading frame 50 332KIAA0746 2.8 KIAA0746 protein 333 PIK3CB 2.8 phosphoinositide-3-kinase,catalytic, beta polypeptide 334 KIAA1841 2.8 KIAA1841 335 PGAP1 2.8 GPIdeacylase 336 KLHL7 2.8 kelch-like 7 (Drosophila) 337 C5orf34 2.8chromosome 5 open reading frame 34 338 CARD8 2.8 caspase recruitmentdomain family, member 8 339 PEG3 2.8 paternally expressed 3 340 ACPL22.8 acid phosphatase-like 2 341 PRR6 2.8 proline rich 6 342 HLA-DQB1 2.8major histocompatibility complex, class II, DQ beta 1 343 TXNDC17 2.8thioredoxin domain containing 17 344 SRI 2.8 sorcin 345 BNIP3 2.8BCL2/adenovirus E1B 19 kDa interacting protein 3 346 AHR 2.8 arylhydrocarbon receptor 347 SPG3A 2.8 spastic paraplegia 3A (autosomaldominant) 348 GSTO1 2.8 glutathione S-transferase omega 1 349 FLI1 2.8Friend leukemia virus integration 1 350 NEK2 2.8 NIMA (never in mitosisgene a)-related kinase 2 351 FLOT1 2.7 flotillin 1 352 FLJ39739 2.7hypothetical FLJ39739 353 LOC728855 2.7 hypothetical protein LOC728855354 EHBP1 2.7 EH domain binding protein 1 355 PAAF1 2.7 proteasomalATPase-associated factor 1 356 HLTF 2.7 helicase-like transcriptionfactor 357 TUFT1 2.7 tuftelin 1 358 TNFRSF11B 2.7 tumor necrosis factorreceptor super- family, member 11b (osteoprotegerin) 359 MMD 2.7monocyte to macrophage differentiation- associated 360 HPS3 2.7Hermansky-Pudlak syndrome 3 361 P2RX5 2.7 purinergic receptor P2X,ligand-gated ion channel, 5 362 C15orf48 2.7 chromosome 15 open readingframe 48 363 ACY1L2 2.7 aminoacylase 1-like 2 364 SFXN3 2.7 sideroflexin3 365 FAS 2.7 Fas (TNF receptor superfamily, member 6) 366 ANAPC10 2.7anaphase promoting complex subunit 10 367 ZNF652 2.7 zinc finger protein652 368 LOXL3 2.7 lysyl oxidase-like 3 369 SLC16A2 2.7 solute carrierfamily 16, member 2 (monocarboxylic acid transporter 8) 370 AMPD3 2.7adenosine monophosphate deaminase (isoform E) 371 CDK5 2.7cyclin-dependent kinase 5 372 PPP3CB 2.7 protein phosphatase 3 (formerly2B), catalytic subunit, beta isoform 373 C9orf30 2.7 chromosome 9 openreading frame 30 374 HLA-DRA 2.7 major histocompatibility complex, classII, DR alpha 375 CAPN2 2.7 calpain 2, (m/II) large subunit 376 MGAT4A2.7 mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A 377 UROD 2.7uroporphyrinogen decarboxylase 378 TNFRSF11A 2.7 tumor necrosis factorreceptor super- family, member 11a, NFKB activator 379 LOC440295 2.7hypothetical protein LOC440295 380 DOCK4 2.7 dedicator of cytokinesis 4381 C6orf145 2.7 chromosome 6 open reading frame 145 382 BCL11B 2.7B-cell CLL/lymphoma 11B (zinc finger protein) 383 RP11- 2.7 deleted in amouse model of primary 529I10.4 ciliary dyskinesia 384 DAZL 2.7 deletedin azoospermia-like 385 CCDC92 2.7 coiled-coil domain containing 92 386LOC389203 2.7 hypothetical gene supported by BC032431 387 FUCA1 2.7fucosidase, alpha-L-1, tissue 388 MGC16169 2.7 hypothetical proteinMGC16169 389 CLDN12 2.7 claudin 12 390 MAK 2.7 male germ cell-associatedkinase 391 XRCC6BP1 2.7 XRCC6 binding protein 1 392 BAD 2.7BCL2-antagonist of cell death 393 MAP9 2.7 microtubule-associatedprotein 9 394 CETN3 2.7 centrin, EF-hand protein, 3 (CDC31 homolog,yeast) 395 CACYBP 2.7 calcyclin binding protein 396 ROBO1 2.7roundabout, axon guidance receptor, homolog 1 (Drosophila) 397 TAX1BP32.6 Tax1 (human T-cell leukemia virus type I) binding protein 3 398FLJ11151 2.6 hypothetical protein FLJ11151 399 ITGA6 2.6 integrin, alpha6 400 RAVER2 2.6 ribonucleoprotein, PTB-binding 2 401 GPR155 2.6 Gprotein-coupled receptor 155 402 SLC8A1 2.6 solute carrier family 8(sodium/calcium exchanger), member 1 403 ABHD7 2.6 abhydrolase domaincontaining 7 404 SYNGR3 2.6 synaptogyrin 3 405 FBXO31 2.6 F-box protein31 406 GK5 2.6 glycerol kinase 5 (putative) 407 TEAD1 2.6 TEA domainfamily member 1 (SV40 transcriptional enhancer factor) 408 CORO1B 2.6coronin, actin binding protein, 1B 409 OSGEPL1 2.6 O-sialoglycoproteinendopeptidase-like 1 410 ACAA2 2.6 acetyl-Coenzyme A acyltransferase 2(mitochondrial 3-oxoacyl-Coenzyme A thiolase) 411 RBMS1 2.6 RNA bindingmotif, single stranded interacting protein 1 412 GM2A 2.6 GM2ganglioside activator 413 COX11 2.6 COX11 homolog, cytochrome c oxidaseassembly protein (yeast) 414 ATPBD4 2.6 ATP binding domain 4 415 GSTM32.6 glutathione S-transferase M3 (brain) 416 SKP2 2.6 S-phasekinase-associated protein 2 (p45) 417 PAK1 2.6 p21/Cdc42/Rac1-activatedkinase 1 (STE20 homolog, yeast) 418 ACTN1 2.6 actinin, alpha 1 419 MYEF22.6 myelin expression factor 2 420 ERLIN2 2.6 ER lipid raft associated 2421 VLDLR 2.6 very low density lipoprotein receptor 422 WDR54 2.6 WDrepeat domain 54 423 GRK5 2.6 G protein-coupled receptor kinase 5 424ARHGAP30 2.6 Rho GTPase activating protein 30 425 ANKRD57 2.6 ankyrinrepeat domain 57 426 MYH10 2.6 myosin, heavy chain 10, non-muscle 427TCEAL1 2.6 transcription elongation factor A (SII)- like 1 428 GALNT122.6 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N-acetylgalactosaminyltransferase 12 (GalNAc-T12) 429 CCDC5 2.6coiled-coil domain containing 5 (spindle associated) 430 ITPR2 2.6inositol 1,4,5-triphosphate receptor, type 2 431 LGALS8 2.6 lectin,galactoside-binding, soluble, 8 (galectin 8) 432 GNB5 2.6 guaninenucleotide binding protein (G protein), beta 5 433 KBTBD11 2.5 kelchrepeat and BTB (POZ) domain containing 11 434 ZDHHC4 2.5 zinc finger,DHHC-type containing 4 435 FREQ 2.5 frequenin homolog (Drosophila) 436ETHE1 2.5 ethylmalonic encephalopathy 1 437 IGFBP2 2.5 insulin-likegrowth factor binding protein 2, 36 kDa 438 CCDC4 2.5 coiled-coil domaincontaining 4 439 PYGL 2.5 phosphorylase, glycogen; liver (Hers disease,glycogen storage disease type VI) 440 TMEM38B 2.5 transmembrane protein38B 441 THEM2 2.5 thioesterase superfamily member 2 442 HRH4 2.5histamine receptor H4 443 C21orf96 2.5 chromosome 21 open reading frame96 444 C11orf49 2.5 chromosome 11 open reading frame 49 445 EIF2C1 2.5eukaryotic translation initiation factor 2C, 1 446 GLB1L3 2.5galactosidase, beta 1 like 3 447 PAFAH1B3 2.5 platelet-activating factoracetylhydrolase, isoform Ib, gamma subunit 29 kDa 448 PHF19 2.5 PHDfinger protein 19 449 TBL1XR1 2.5 transducin (beta)-like 1X-linkedreceptor 1 450 SORD 2.5 sorbitol dehydrogenase 451 MND1 2.5 meioticnuclear divisions 1 homolog (S. cerevisiae) 452 MSRB2 2.5 methioninesulfoxide reductase B2 453 ZNF169 2.5 zinc finger protein 169 454 LANCL12.5 LanC lantibiotic synthetase component C- like 1 (bacterial) 455KLHL6 2.5 kelch-like 6 (Drosophila) 456 MYO5A 2.5 myosin VA (heavy chain12, myoxin) 457 RPS27L 2.5 ribosomal protein S27-like 458 LOC440288 2.5similar to FLJ16518 protein 459 IVNS1ABP 2.5 influenza virus NS1Abinding protein 460 SLC39A4 2.5 solute carrier family 39 (zinctransporter), member 4 461 DMC1 2.5 DMC1 dosage suppressor of mck1homolog, meiosis-specific homologous recombination (yeast) 462 RAI14 2.5retinoic acid induced 14 463 DPYSL2 2.5 dihydropyrimidinase-like 2 464HEATR2 2.5 HEAT repeat containing 2 465 FSD1 2.5 fibronectin type IIIand SPRY domain containing 1 466 GCHFR 2.5 GTP cyclohydrolase I feedbackregulator 467 ESD 2.5 esterase D/formylglutathione hydrolase 468 RAD54B2.5 RAD54 homolog B (S. cerevisiae) 469 CPNE2 2.5 copine II 470 ARHGEF122.5 Rho guanine nucleotide exchange factor (GEF) 12 471 RECK 2.5reversion-inducing-cysteine-rich protein with kazal motifs 472 AK3L1 2.5adenylate kinase 3-like 1 473 SGCB 2.5 sarcoglycan, beta (43 kDadystrophin- associated glycoprotein) 474 COL6A2 2.5 collagen, type VI,alpha 2 475 RNASEH2A 2.5 ribonuclease H2, subunit A 476 CRTAP 2.5cartilage associated protein 477 PRMT2 2.5 protein argininemethyltransferase 2 478 IFT81 2.5 intraflagellar transport 81 homolog(Chlamydomonas) 479 SKAP2 2.5 src kinase associated phosphoprotein 2 480NT5C3L 2.5 5′-nucleotidase, cytosolic III-like 481 DIP2B 2.5 DIP2disco-interacting protein 2 homolog B (Drosophila) 482 LOC730846 2.5similar to S-adenosylmethionine decarboxylase proenzyme 2 (AdoMetDC 2)(SamDC 2) 483 C4orf34 2.5 chromosome 4 open reading frame 34 484 IKZF42.5 IKAROS family zinc finger 4 (Eos) 485 COMT 2.5catechol-O-methyltransferase 486 ECOP 2.5 EGFR-coamplified andoverexpressed protein 487 KIAA1715 2.5 KIAA1715 488 RRAGC 2.5Ras-related GTP binding C 489 C8orf70 2.5 chromosome 8 open readingframe 70 490 LRFN3 2.5 leucine rich repeat and fibronectin type IIIdomain containing 3 491 LOC642236 2.5 similar to FRG1 protein (FSHDregion gene 1 protein) 492 ITGA3 2.4 integrin, alpha 3 (antigen CD49C,alpha 3 subunit of VLA-3 receptor) 493 FVT1 2.4 follicular lymphomavariant translocation 1 494 HSPB1 2.4 heat shock 27 kDa protein 1 495BMP2K 2.4 BMP2 inducible kinase 496 SNRPN 2.4 small nuclearribonucleoprotein polypeptide N 497 TDRKH 2.4 tudor and KH domaincontaining 498 RAB7L1 2.4 RAB7, member RAS oncogene family- like 1 499TEAD4 2.4 TEA domain family member 4 500 SERPINB6 2.4 serpin peptidaseinhibitor, clade B (ovalbumin), member 6 501 GTF2A1 2.4 generaltranscription factor IIA, 1, 19/37 kDa 502 CCDC15 2.4 coiled-coil domaincontaining 15 503 ALS2CR4 2.4 amyotrophic lateral sclerosis 2 (juvenile)chromosome region, candidate 4 504 DLG7 2.4 discs, large homolog 7(Drosophila) 505 ZSWIM7 2.4 zinc finger, SWIM-type containing 7 506 EHD32.4 EH-domain containing 3 507 ANG 2.4 angiogenin, ribonuclease, RNase Afamily, 5 508 HSD17B6 2.4 hydroxysteroid (17-beta) dehydrogenase 6homolog (mouse) 509 CISD3 2.4 CDGSH iron sulfur domain 3 510 CBR4 2.4carbonyl reductase 4 511 ORC5L 2.4 origin recognition complex, subunit5-like (yeast) 512 POLR3G 2.4 polymerase (RNA) III (DNA directed)polypeptide G (32 kD) 513 LASP1 2.4 LIM and SH3 protein 1 514 XPNPEP32.4 X-prolyl aminopeptidase (aminopeptidase P) 3, putative 515 NLRC3 2.4NLR family, CARD domain containing 3 516 LOC133874 2.4 hypothetical geneLOC133874 517 TMEM173 2.4 transmembrane protein 173 518 COG6 2.4component of oligomeric golgi complex 6 519 TNFSF13B 2.4 tumor necrosisfactor (ligand) superfamily, member 13b 520 CUEDC2 2.4 CUE domaincontaining 2 521 PSD3 2.4 pleckstrin and Sec7 domain containing 3 522SCRN1 2.4 secernin 1 523 SCOC 2.4 short coiled-coil protein 524 FAM45A2.4 family with sequence similarity 45, member A 525 TOM1L1 2.4 targetof myb1 (chicken)-like 1 526 PRIM2 2.4 primase, DNA, polypeptide 2 (58kDa) 527 SMTN 2.4 smoothelin 528 TMEM50B 2.4 transmembrane protein 50B529 THEM5 2.4 thioesterase superfamily member 5 530 KIAA0146 2.4KIAA0146 531 COMMD8 2.4 COMM domain containing 8 532 KIF20A 2.4 kinesinfamily member 20A 533 NEGR1 2.4 neuronal growth regulator 1 534 KLF7 2.4Kruppel-like factor 7 (ubiquitous) 535 C1orf93 2.4 chromosome 1 openreading frame 93 536 KCTD15 2.4 potassium channel tetramerisation domaincontaining 15 537 LACTB2 2.4 lactamase, beta 2 538 TCEA2 2.4transcription elongation factor A (SII), 2 539 GLRX 2.4 glutaredoxin(thioltransferase) 540 KIAA1804 2.4 mixed lineage kinase 4 541 ATP2C12.4 ATPase, Ca++ transporting, type 2C, member 1 542 LOC339803 2.4hypothetical protein LOC339803 543 NDUFA8 2.4 NADH dehydrogenase(ubiquinone) 1 alpha subcomplex, 8, 19 kDa 544 ADA 2.4 adenosinedeaminase 545 SLC25A4 2.4 solute carrier family 25 (mitochondrialcarrier; adenine nucleotide translocator), member 4 546 IDH1 2.4isocitrate dehydrogenase 1 (NADP+), soluble 547 TANC2 2.4tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2548 AGL 2.4 amylo-1, 6-glucosidase, 4-alpha-glucano- transferase(glycogen debranching enzyme, glycogen storage disease type III) 549MAGED2 2.4 melanoma antigen family D, 2 550 JUB 2.4 jub, ajuba homolog(Xenopus laevis) 551 BCAT1 2.4 branched chain aminotransferase 1,cytosolic 552 IRAK1BP1 2.4 interleukin-1 receptor-associated kinase 1binding protein 1 553 RAB6IP1 2.4 RAB6 interacting protein 1 554 MOCS22.4 molybdenum cofactor synthesis 2 555 DMRT1 2.4 doublesex and mab-3related transcription factor 1 556 HLA-DRB1 2.4 major histocompatibilitycomplex, class II, DR beta 1 557 GLRX2 2.4 glutaredoxin 2 558 IL11 2.4interleukin 11 559 FOXA1 2.4 forkhead box A1 560 SLC39A10 2.4 solutecarrier family 39 (zinc transporter), member 10 561 SNX24 2.4 sortingnexin 24 562 NGFRAP1L1 2.4 NGFRAP1-like 1 563 C9orf64 2.4 chromosome 9open reading frame 64 564 FAM82B 2.4 family with sequence similarity 82,member B 565 C1orf41 2.4 chromosome 1 open reading frame 41 566 TMED82.4 transmembrane emp24 protein transport domain containing 8 567 TLOC12.4 translocation protein 1 568 GPX1 2.4 glutathione peroxidase 1 569GPR157 2.4 G protein-coupled receptor 157 570 MGC61571 2.4 hypotheticalprotein MGC61571 571 RNF14 2.4 ring finger protein 14 572 SLC41A1 2.4solute carrier family 41, member 1 573 CAMSAP1L1 2.4 calmodulinregulated spectrin-associated protein 1-like 1 574 HS2ST1 2.4 heparansulfate 2-O-sulfotransferase 1 575 SNX3 2.4 sorting nexin 3 576 STK392.3 serine threonine kinase 39 (STE20/SPS1 homolog, yeast) 577 GRSF1 2.3G-rich RNA sequence binding factor 1 578 TXNDC5 2.3 thioredoxin domaincontaining 5 579 PDLIM7 2.3 PDZ and LIM domain 7 (enigma) 580 LOC7301072.3 similar to Glycine cleavage system H protein, mitochondrialprecursor 581 PKP4 2.3 plakophilin 4 582 PHGDH 2.3 phosphoglyceratedehydrogenase 583 RASGRP4 2.3 RAS guanyl releasing protein 4 584 C3orf632.3 chromosome 3 open reading frame 63 585 MTX2 2.3 metaxin 2 586 CDC202.3 cell division cycle 20 homolog (S. cerevisiae) 587 ATP6V1A 2.3ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A 588 SCN3A 2.3sodium channel, voltage-gated, type III, alpha subunit 589 MICALL1 2.3MICAL-like 1 590 C1orf26 2.3 chromosome 1 open reading frame 26 591ZCRB1 2.3 zinc finger CCHC-type and RNA binding motif 1 592 ATG4C 2.3ATG4 autophagy related 4 homolog C (S. cerevisiae) 593 EIF2AK4 2.3eukaryotic translation initiation factor 2 alpha kinase 4 594 FLJ325492.3 hypothetical protein FLJ32549 595 PPID 2.3 peptidylprolyl isomeraseD (cyclophilin D) 596 SCCPDH 2.3 saccharopine dehydrogenase (putative)597 ICMT 2.3 isoprenylcysteine carboxyl methyltransferase 598 CLIP1 2.3CAP-GLY domain containing linker protein 1 599 ENY2 2.3 enhancer ofyellow 2 homolog (Drosophila) 600 RABL5 2.3 RAB, member RAS oncogenefamily-like 5 601 KIAA1430 2.3 KIAA1430 602 BCL6 2.3 B-cell CLL/lymphoma6 (zinc finger protein 51) 603 GBAS 2.3 glioblastoma amplified sequence604 COQ3 2.3 coenzyme Q3 homolog, methyltransferase (S. cerevisiae) 605ATG10 2.3 ATG10 autophagy related 10 homolog (S. cerevisiae) 606KIAA1211 2.3 KIAA1211 protein 607 SPA17 2.3 sperm autoantigenic protein17 608 EIF2C4 2.3 eukaryotic translation initiation factor 2C, 4 609PECI 2.3 peroxisomal D3,D2-enoyl-CoA isomerase 610 JAKMIP2 2.3 januskinase and microtubule interacting protein 2 611 HEBP1 2.3 heme bindingprotein 1 612 NCOA1 2.3 nuclear receptor coactivator 1 613 PHCA 2.3phytoceramidase, alkaline 614 B3GNT1 2.3 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 615 FARS2 2.3 phenylalanyl-tRNAsynthetase 2, mitochondrial 616 CEBPD 2.3 CCAAT/enhancer binding protein(C/EBP), delta 617 ACBD5 2.3 acyl-Coenzyme A binding domain containing 5618 AMZ2 2.3 archaemetzincins-2 619 DPH4 2.3 DPH4, JJJ3 homolog (S.cerevisiae) 620 RAB32 2.3 RAB32, member RAS oncogene family 621 WDFY32.3 WD repeat and FYVE domain containing 3 622 CCDC91 2.3 coiled-coildomain containing 91 623 ALDOC 2.3 aldolase C, fructose-bisphosphate 624HADH 2.3 hydroxyacyl-Coenzyme A dehydrogenase 625 GTF2IRD1 2.3 GTF2Irepeat domain containing 1 626 ATF3 2.3 activating transcription factor3 627 CENTB1 2.3 centaurin, beta 1 628 ROR1 2.3 receptor tyrosinekinase-like orphan receptor 1 629 JHDM1D 2.3 jumonji C domain-containinghistone demethylase 1 homolog D (S. cerevisiae) 630 STYXL1 2.3serine/threonine/tyrosine interacting-like 1 631 SPAG16 2.3 spermassociated antigen 16 632 LOC153364 2.3 similar tometallo-beta-lactamase superfamily protein 633 RPL23AP7 2.3 ribosomalprotein L23a pseudogene 7 634 SEPT9 2.3 septin 9 635 HEBP2 2.3 hemebinding protein 2 636 FAIM 2.3 Fas apoptotic inhibitory molecule 637NUDT7 2.3 nudix (nucleoside diphosphate linked moiety X)-type motif 7638 HSPA2 2.3 heat shock 70 kDa protein 2 639 ACSS2 2.3 acyl-CoAsynthetase short-chain family member 2 640 PCTP 2.3 phosphatidylcholinetransfer protein 641 SH3BP5 2.3 SH3-domain binding protein 5 (BTK-associated) 642 ABCG1 2.3 ATP-binding cassette, sub-family G (WHITE),member 1 643 IHPK2 2.3 inositol hexaphosphate kinase 2 644 ZFYVE21 2.3zinc finger, FYVE domain containing 21 645 PBK 2.3 PDZ binding kinase646 BAX 2.3 BCL2-associated X protein 647 C4orf27 2.3 chromosome 4 openreading frame 27 648 C9orf46 2.3 chromosome 9 open reading frame 46 649NMRAL1 2.3 NmrA-like family domain containing 1 650 NDUFB10 2.3 NADHdehydrogenase (ubiquinone) 1 beta subcomplex, 10, 22 kDa 651 NT5DC2 2.35′-nucleotidase domain containing 2 652 ZDHHC2 2.3 zinc finger,DHHC-type containing 2 653 MIF4GD 2.3 MIF4G domain containing 654 IMPDH22.2 IMP (inosine monophosphate) dehydrogenase 2 655 C1RL 2.2 complementcomponent 1, r subcomponent-like 656 ALS2CR2 2.2 amyotrophic lateralsclerosis 2 (juvenile) chromosomeregion, candidate 2 657 VAV2 2.2 vav 2guanine nucleotide exchange factor 658 VPS8 2.2 vacuolar protein sorting8 homolog (S. cerevisiae) 659 NKIRAS1 2.2 NFKB inhibitor interactingRas-like 1 660 ARL3 2.2 ADP-ribosylation factor-like 3 661 FAM129A 2.2family with sequence similarity 129, member A 662 RPS6KA2 2.2 ribosomalprotein S6 kinase, 90 kDa, polypeptide 2 663 RAB31 2.2 RAB31, member RASoncogene family 664 ADCK2 2.2 aarF domain containing kinase 2 665 GPD22.2 glycerol-3-phosphate dehydrogenase 2 (mitochondrial) 666 TUBB6 2.2tubulin, beta 6 667 PARVG 2.2 parvin, gamma 668 LRP10 2.2 low densitylipoprotein receptor-related protein 10 669 MED25 2.2 mediator complexsubunit 25 670 MVP 2.2 major vault protein 671 LCP2 2.2 lymphocytecytosolic protein 2 (SH2 domaincontaining leukocyte protein of 76 kDa)672 C17orf75 2.2 chromosome 17 open reading frame 75 673 LIX1L 2.2 Lix1homolog (mouse)-like 674 C3orf64 2.2 chromosome 3 open reading frame 64675 RCCD1 2.2 RCC1 domain containing 1 676 TMEM55A 2.2 transmembraneprotein 55A 677 CPNE3 2.2 copine III 678 FUNDC1 2.2 FUN14 domaincontaining 1 679 MAP3K9 2.2 mitogen-activated protein kinase kinasekinase 9 680 C5orf40 2.2 chromosome 5 open reading frame 40 681 CPT2 2.2carnitine palmitoyltransferase II 682 H1F0 2.2 H1 histone family, member0 683 TADA3L 2.2 transcriptional adaptor 3 (NGG1 homolog, yeast)-like684 HLA-DMB 2.2 major histocompatibility complex, class II, DM beta 685DAPP1 2.2 dual adaptor of phosphotyrosine and 3- phosphoinositides 686HSD17B4 2.2 hydroxysteroid (17-beta) dehydrogenase 4 687 LGALS1 2.2lectin, galactoside-binding, soluble, 1 (galectin 1) 688 NARF 2.2nuclear prelamin A recognition factor 689 ANKRA2 2.2 ankyrin repeat,family A (RFXANK- like), 2 690 SNX10 2.2 sorting nexin 10 691 TMCO3 2.2transmembrane and coiled-coil domains 3 692 SLC22A4 2.2 solute carrierfamily 22 (organic cation transporter), member 4 693 CMTM7 2.2 CKLF-likeMARVEL transmembrane domain containing 7 694 SGPP1 2.2sphingosine-1-phosphate phosphatase 1 695 SV2A 2.2 synaptic vesicleglycoprotein 2A 696 SEC22C 2.2 SEC22 vesicle trafficking protein homologC (S. cerevisiae) 697 ELMOD2 2.2 ELMO/CED-12 domain containing 2 698FLJ35348 2.2 FLJ35348 699 RWDD3 2.2 RWD domain containing 3 700 TXNIP2.2 thioredoxin interacting protein 701 GMPR2 2.2 guanosinemonophosphate reductase 2 702 RPGRIP1L 2.2 RPGRIP1-like 703 EXOD1 2.2exonuclease domain containing 1 704 RNF135 2.2 ring finger protein 135705 FECH 2.2 ferrochelatase (protoporphyria) 706 COL6A1 2.2 collagen,type VI, alpha 1 707 C2orf28 2.2 chromosome 2 open reading frame 28 708MTPN 2.2 myotrophin 709 TTC7A 2.2 tetratricopeptide repeat domain 7A 710CPOX 2.2 coproporphyrinogen oxidase 711 SCAMP1 2.2 secretory carriermembrane protein 1 712 C11orf17 2.2 chromosome 11 open reading frame 17713 FAM125A 2.2 family with sequence similarity 125, member A 714FAM134B 2.2 family with sequence similarity 134, member B 715 LOC2835512.2 hypothetical protein LOC283551 716 CA12 2.2 carbonic anhydrase XII717 ATPAF1 2.2 ATP synthase mitochondrial F1 complex assembly factor 1718 TBC1D5 2.2 TBC1 domain family, member 5 719 CPNE8 2.2 copine VIII720 ADD2 2.2 adducin 2 (beta) 721 FLJ43663 2.2 hypothetical proteinFLJ43663 722 SLC1A4 2.2 solute carrier family 1 (glutamate/neutral aminoacid transporter), member 4 723 MKL2 2.2 MKL/myocardin-like 2 724 MUM12.2 melanoma associated antigen (mutated) 1 725 FBXL16 2.2 F-box andleucine-rich repeat protein 16 726 LAPTM4B 2.2 lysosomal associatedprotein transmembrane 4 beta 727 HCFC1R1 2.2 host cell factor C1regulator 1 (XPO1 dependent) 728 CYFIP1 2.2 cytoplasmic FMR1 interactingprotein 1 729 STXBP1 2.2 syntaxin binding protein 1 730 SLC25A12 2.2solute carrier family 25 (mitochondrial carrier, Aralar), member 12 731TPM1 2.2 tropomyosin 1 (alpha) 732 PIGP 2.2 phosphatidylinositol glycananchor biosynthesis, class P 733 PAWR 2.2 PRKC, apoptosis, WT1,regulator 734 CSK 2.2 c-src tyrosine kinase 735 HRSP12 2.2heat-responsive protein 12 736 C20orf23 2.2 chromosome 20 open readingframe 23 737 GSTM4 2.2 glutathione S-transferase M4 738 ARL2 2.2ADP-ribosylation factor-like 2 739 ADK 2.2 adenosine kinase 740 RABAC12.2 Rab acceptor 1 (prenylated) 741 DEPDC1B 2.2 DEP domain containing 1B742 FSCN1 2.1 fascin homolog 1, actin-bundling protein(Strongylocentrotus purpuratus) 743 IRF2BP2 2.1 interferon regulatoryfactor 2 binding protein 2 744 EIF4EBP1 2.1 eukaryotic translationinitiation factor 4E binding protein 1 745 RFTN1 2.1 raftlin, lipid raftlinker 1 746 C6orf32 2.1 chromosome 6 open reading frame 32 747 SAV1 2.1salvador homolog 1 (Drosophila) 748 RCBTB1 2.1 regulator of chromosomecondensation (RCC1) and BTB (POZ) domain containing protein 1 749 AS3MT2.1 arsenic (+3 oxidation state) methyltransferase 750 PROS1 2.1 proteinS (alpha) 751 LOC339804 2.1 hypothetical gene supported by AK075484;BC014578 752 ACADSB 2.1 acyl-Coenzyme A dehydrogenase, short/branchedchain 753 PLAUR 2.1 plasminogen activator, urokinase receptor 754 HERC42.1 hect domain and RLD 4 755 SLAIN1 2.1 SLAIN motif family, member 1756 C9orf119 2.1 chromosome 9 open reading frame 119 757 PAQR8 2.1progestin and adipoQ receptor family member VIII 758 DARS 2.1aspartyl-tRNA synthetase 759 ANKRD22 2.1 ankyrin repeat domain 22 760UROS 2.1 uroporphyrinogen III synthase (congenital erythropoieticporphyria) 761 TNRC6B 2.1 trinucleotide repeat containing 6B 762 NDRG32.1 NDRG family member 3 763 LYRM7 2.1 Lyrm7 homolog (mouse) 764TNFRSF25 2.1 tumor necrosis factor receptor superfamily, member25 765CNKSR3 2.1 CNKSR family member 3 766 INPP5F 2.1 inositolpolyphosphate-5-phosphatase F 767 TNS1 2.1 tensin 1 768 TTC32 2.1tetratricopeptide repeat domain 32 769 SAMD1 2.1 sterile alpha motifdomain containing 1 770 ARHGAP5 2.1 Rho GTPase activating protein 5 771MAP3K13 2.1 mitogen-activated protein kinase kinase kinase 13 772 PGM22.1 phosphoglucomutase 2 773 COX18 2.1 COX18 cytochrome c oxidaseassembly homolog (S. cerevisiae) 774 LOC339751 2.1 hypothetical proteinLOC339751 775 BLVRB 2.1 biliverdin reductase B (flavin reductase(NADPH)) 776 CKB 2.1 creatine kinase, brain 777 PHKB 2.1 phosphorylasekinase, beta 778 M6PRBP1 2.1 mannose-6-phosphate receptor bindingprotein 1 779 SEC61A2 2.1 Sec61 alpha 2 subunit (S. cerevisiae) 780 HMMR2.1 hyaluronan-mediated motility receptor (RHAMM) 781 PPP1R7 2.1 proteinphosphatase 1, regulatory (inhibitor) subunit 7 782 YIPF1 2.1 Yip1domain family, member 1 783 PHF15 2.1 PHD finger protein 15 784 C6orf2112.1 chromosome 6 open reading frame 211 785 OAT 2.1 ornithineaminotransferase (gyrate atrophy) 786 HLA-DRB5 2.1 majorhistocompatibility complex, class II, DR beta 5 787 DYNC1H1 2.1 dynein,cytoplasmic 1, heavy chain 1 788 ITGB3BP 2.1 integrin beta 3 bindingprotein (beta3- endonexin) 789 RABEPK 2.1 Rab9 effector protein withkelch motifs 790 LPIN1 2.1 lipin 1 791 F8 2.1 coagulation factor VIII,procoagulant component (hemophilia A) 792 ARHGAP19 2.1 Rho GTPaseactivating protein 19 793 CCDC90A 2.1 coiled-coil domain containing 90A794 AMMECR1 2.1 Alport syndrome, mental retardation, midface hypoplasiaand elliptocytosis chromosomal region, gene 1 795 KIAA1279 2.1 KIAA1279796 CRYZL1 2.1 crystallin, zeta (quinone reductase)-like 1 797 HNRNPU2.1 heterogeneous nuclear ribonucleoprotein U (scaffold attachmentfactor A) 798 PRKAR2A 2.1 protein kinase, cAMP-dependent, regulatory,type II, alpha 799 CASK 2.1 calcium/calmodulin-dependent serine proteinkinase (MAGUK family) 800 EFCAB4B 2.1 EF-hand calcium binding domain 4B801 RDX 2.1 radixin 802 BLR1 2.1 Burkitt lymphoma receptor 1, GTPbinding protein (chemokine (C—X—C motif) receptor 5) 803 TFEB 2.1transcription factor EB 804 RTN4IP1 2.1 reticulon 4 interacting protein1 805 C13orf21 2.1 chromosome 13 open reading frame 21 806 SCFD2 2.1sec1 family domain containing 2 807 COG5 2.1 component of oligomericgolgi complex 5 808 TST 2.1 thiosulfate sulfurtransferase (rhodanese)809 DNAJC13 2.1 DnaJ (Hsp40) homolog, subfamily C, member 13 810 CCNB22.1 cyclin B2 811 L1CAM 2.1 L1 cell adhesion molecule 812 DEF6 2.1differentially expressed in FDCP 6 homolog (mouse) 813 TNFRSF19 2.1tumor necrosis factor receptor superfamily, member 19 814 PTPLA 2.1protein tyrosine phosphatase-like (proline instead of catalyticarginine), member A 815 DGKI 2.1 diacylglycerol kinase, iota 816 SPIN42.1 spindlin family, member 4 817 GBE1 2.1 glucan (1,4-alpha-),branching enzyme 1 (glycogen branching enzyme, Andersen disease,glycogen storage disease type IV) 818 PTPN12 2.1 protein tyrosinephosphatase, non- receptor type 12 819 CFDP1 2.1 craniofacialdevelopment protein 1 820 C14orf100 2.1 chromosome 14 open reading frame100 821 SFN 2.1 stratifin 822 GCSH 2.1 glycine cleavage system protein H(aminomethyl carrier) 823 PTP4A2 2.1 protein tyrosine phosphatase typeIVA, member 2 824 HMG20B 2.1 high-mobility group 20B 825 SMAD7 2.1 SMADfamily member 7 826 ACYP1 2.1 acylphosphatase 1, erythrocyte (common)type 827 HIBCH 2.1 3-hydroxyisobutyryl-Coenzyme A hydrolase 828 ART3 2.1ADP-ribosyltransferase 3 829 SH3YL1 2.1 SH3 domain containing,Ysc84-like 1 (S. cerevisiae) 830 ADFP 2.1 adiposedifferentiation-related protein 831 HDAC9 2.1 histone deacetylase 9 832CTTNBP2NL 2.1 CTTNBP2 N-terminal like 833 RNASEH2B 2.1 ribonuclease H2,subunit B 834 LCK 2.1 lymphocyte-specific protein tyrosine kinase 835KCTD12 2.1 potassium channel tetramerisation domain containing 12 836PCYOX1 2.1 prenylcysteine oxidase 1 837 TCEAL3 2.1 transcriptionelongation factor A (SII)- like 3 838 PAQR3 2.1 progestin and adipoQreceptor family member III 839 TBC1D10C 2.1 TBC1 domain family, member10C 840 SNX30 2.1 sorting nexin family member 30 841 PHKA1 2.1phosphorylase kinase, alpha 1 (muscle) 842 HLA-DRB4 2.1 majorhistocompatibility complex, class II, DR beta 4 843 GALNT6 2.1UDP-N-acetyl-alpha-D- galactosamine:polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) 844 TCF12 2.1transcription factor 12 (HTF4, helix-loop- helix transcription factors4) 845 CHST3 2.1 carbohydrate (chondroitin 6) sulfotransferase 3 846TUBB3 2.1 tubulin, beta 3 847 AURKA 2.1 aurora kinase A 848 MAD2L2 2.1MAD2 mitotic arrest deficient-like 2 (yeast) 849 CTDSP2 2.1 CTD(carboxy-terminal domain, RNA polymerase II, polypeptide A) smallphosphatase 2 850 COQ5 2.1 coenzyme Q5 homolog, methyltransferase (S.cerevisiae) 851 FAM21C 2.1 family with sequence similarity 21, member C852 C6orf57 2.1 chromosome 6 open reading frame 57 853 UNQ501 2.1MBC3205 854 IL27RA 2.1 interleukin 27 receptor, alpha 855 KCTD3 2.1potassium channel tetramerisation domain containing 3 856 DERA 2.12-deoxyribose-5-phosphate aldolase homolog (C. elegans) 857 ANXA4 2.1annexin A4 858 CCDC104 2.1 coiled-coil domain containing 104 859 VDR 2.1vitamin D (1,25-dihydroxyvitamin D3) receptor 860 POMZP3 2.1 POM (POM121homolog, rat) and ZP3 fusion 861 PQLC3 2.1 PQ loop repeat containing 3862 SCRN3 2.1 secernin 3 863 FZD7 2.1 frizzled homolog 7 (Drosophila)864 LOC145842 2.1 hypothetical protein LOC145842 865 C11orf73 2.1chromosome 11 open reading frame 73 866 C20orf19 2.1 chromosome 20 openreading frame 19 867 MORC4 2.1 MORC family CW-type zinc finger 4 868NAGA 2.1 N-acetylgalactosaminidase, alpha- 869 APLP2 2.1 amyloid beta(A4) precursor-like protein 2 870 RHOQ 2.1 ras homolog gene family,member Q 871 BCS1L 2.1 BCS1-like (yeast) 872 C10orf4 2.1 chromosome 10open reading frame 4 873 ADCY7 2.1 adenylate cyclase 7 874 FAM21A 2.1family with sequence similarity 21, member A 875 CYB5B 2.1 cytochrome b5type B (outer mitochondrial membrane) 876 TTC3 2.0 tetratricopeptiderepeat domain 3 877 CCDC111 2.0 coiled-coil domain containing 111 878ABHD14A 2.0 abhydrolase domain containing 14A 879 MAGED1 2.0 melanomaantigen family D, 1 880 MEN1 2.0 multiple endocrine neoplasia I 881MRPL40 2.0 mitochondrial ribosomal protein L40 882 CNOT6L 2.0 CCR4-NOTtranscription complex, subunit 6-like 883 CCDC82 2.0 coiled-coil domaincontaining 82 884 FUT7 2.0 fucosyltransferase 7 (alpha (1,3)fucosyltransferase) 885 NEK6 2.0 NIMA (never in mitosis gene a)-relatedkinase 6 886 PSIP1 2.0 PC4 and SFRS1 interacting protein 1 887 CDS1 2.0CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1 888MAPK9 2.0 mitogen-activated protein kinase 9 889 ZC3HAV1L 2.0 zincfinger CCCH-type, antiviral 1-like 890 TMEM19 2.0 transmembrane protein19 891 JMJD2D 2.0 jumonji domain containing 2D 892 FOXP4 2.0 forkheadbox P4 893 PFKM 2.0 phosphofructokinase, muscle 894 DNAJA4 2.0 DnaJ(Hsp40) homolog, subfamily A, member 4 895 MRPL39 2.0 mitochondrialribosomal protein L39 896 XRCC4 2.0 X-ray repair complementing defectiverepair in Chinese hamster cells 4 897 IQCK 2.0 IQ motif containing K 898SH3GLB1 2.0 SH3-domain GRB2-like endophilin B1 899 CCDC52 2.0coiled-coil domain containing 52 900 YES1 2.0 v-yes-1 Yamaguchi sarcomaviral oncogene homolog 1 901 SLC37A1 2.0 solute carrier family 37(glycerol-3- phosphate transporter), member 1 902 STK38L 2.0serine/threonine kinase 38 like 903 SLC2A4RG 2.0 SLC2A4 regulator 904DIAPH3 2.0 diaphanous homolog 3 (Drosophila) 905 CERKL 2.0 ceramidekinase-like 906 MAF 2.0 v-maf musculoaponeurotic fibrosarcoma oncogenehomolog (avian) 907 SNCA 2.0 synuclein, alpha (non A4 component ofamyloid precursor) 908 KCNC4 2.0 potassium voltage-gated channel, Shaw-related subfamily, member 4 909 JAKMIP1 2.0 janus kinase and microtubuleinteracting protein 1 910 CISD2 2.0 CDGSH iron sulfur domain 2 911CCDC90B 2.0 coiled-coil domain containing 90B 912 ING2 2.0 inhibitor ofgrowth family, member 2 913 NPL 2.0 N-acetylneuraminate pyruvate lyase(dihydrodipicolinate synthase) 914 DNAJC10 2.0 DnaJ (Hsp40) homolog,subfamily C, member 10 915 TUBB2C 2.0 tubulin, beta 2C 916 CCNG1 2.0cyclin G1 917 S100PBP 2.0 S100P binding protein 918 SCYL2 2.0 SCY1-like2 (S. cerevisiae) 919 MRPL43 2.0 mitochondrial ribosomal protein L43 920TMEM106C 2.0 transmembrane protein 106C 921 SAT2 2.0 spermidine/spermineN1-acetyltransferase 2 922 TIGD2 2.0 tigger transposable element derived2 923 UEVLD 2.0 UEV and lactate/malate dehyrogenase domains 924 NUBPL2.0 nucleotide binding protein-like 925 GALC 2.0 galactosylceramidase926 HMGA1 2.0 high mobility group AT-hook 1 927 CMAS 2.0 cytidinemonophosphate N- acetylneuraminic acid synthetase 928 S100A11 2.0 S100calcium binding protein A11 929 CTSB 2.0 cathepsin B 930 ASCL1 2.0achaete-scute complex homolog 1 (Drosophila) 931 TNNT1 2.0 troponin Ttype 1 (skeletal, slow) 932 COPG2 2.0 coatomer protein complex, subunitgamma 2 933 GKAP1 2.0 G kinase anchoring protein 1 934 USP13 2.0ubiquitin specific peptidase 13 (isopeptidase T-3) 935 SPTBN1 2.0spectrin, beta, non-erythrocytic 1 936 MGST2 2.0 microsomal glutathioneS-transferase 2 937 PEX1 2.0 peroxisome biogenesis factor 1 938 CPVL 2.0carboxypeptidase, vitellogenic-like 939 LOC729604 2.0 hypotheticalprotein LOC729604 940 LOC26010 2.0 viral DNA polymerase-transactivatedprotein 6 941 ADAM19 2.0 ADAM metallopeptidase domain 19 (meltrin beta)942 SPAST 2.0 spastin 943 PLRG1 2.0 pleiotropic regulator 1 (PRL1homolog, Arabidopsis) 944 LOC647121 2.0 similar to embigin homolog 945CMTM3 2.0 CKLF-like MARVEL transmembrane domain containing 3 946 BUB12.0 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast)

TABLE 4 Human genes that are down-regulated in Th17 conditions. Genesknown to be down-regulated in the Th17 pathway are highlighted in boldas an indication of the validity of the data. fold order gene namechange description 1 IL3 −136.3 interleukin 3 (colony-stimulatingfactor, multiple) 2 IL4 −96.2 interleukin 4 3 INSM1 −91.8insulinoma-associated 1 4 CCL1 −36.0 chemokine (C-C motif) ligand 1 5SPP1 −32.4 secreted phosphoprotein 1 (osteo- pontin, bone sialoproteinI, early T- lymphocyte activation 1) 6 PTGER2 −31.7 prostaglandin Ereceptor 2 (subtype EP2), 53 kDa 7 TNFSF8 −22.7 tumor necrosis factor(ligand) superfamily, member 8 8 CLECL1 −21.9 C-type lectin-like 1 9GATA3 −21.8 GATA binding protein 3 10 NA −20.9 NA 11 XCL2 −20.2chemokine (C motif) ligand 2 12 XCL1 −19.0 chemokine (C motif) ligand 113 SESN3 −18.7 sestrin 3 14 PIP5K1B −16.0phosphatidylinositol-4-phosphate 5- kinase, type I, beta 15 MEF2C −14.7myocyte enhancer factor 2C 16 FOXP1 −14.7 forkhead box P1 17 FGL2 −13.0fibrinogen-like 2 18 KBTBD7 −12.7 kelch repeat and BTB (POZ) domaincontaining 7 19 KCNJ2 −12.7 potassium inwardly-rectifying channel,subfamily J, member 2 20 TMEM46 −12.6 transmembrane protein 46 21 AHI1−11.9 Abelson helper integration site 1 22 FGFBP2 −11.6 fibroblastgrowth factor binding protein 2 23 TNIP3 −11.2 TNFAIP3 interactingprotein 3 24 CD38 −11.2 CD38 molecule 25 PDE7B −11.0 phosphodiesterase7B 26 IL13 −10.7 interleukin 13 27 MAP7 −10.6 microtubule-associatedprotein 7 28 BACH2 −10.6 BTB and CNC homology 1, basic leucine zippertranscription factor 2 29 CSF2 −10.5 colony stimulating factor 2(granulocyte-macrophage) 30 IFNG −10.3 interferon, gamma 31 GBP5 −10.3guanylate binding protein 5 32 RHOBTB3 −10.2 Rho-related BTB domaincontaining 3 33 GBP1 −10.0 guanylate binding protein 1, interferon-inducible, 67 kDa 34 PRSS23 −9.3 protease, serine, 23 35 PLXNC1 −9.3plexin C1 36 PTGS2 −9.1 prostaglandin-endoperoxide synthase 2(prostaglandin G/H synthase and cyclooxygenase) 37 ZEB2 −8.8 zinc fingerE-box binding homeobox 2 38 CTHRC1 −8.5 collagen triple helix repeatcontaining 1 39 APOL6 −8.3 apolipoprotein L, 6 40 TAC1 −8.1 tachykinin,precursor 1 (substance K, substance P, neurokinin 1, neurokinin 2,neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma) 41NRP1 −7.9 neuropilin 1 42 RDH10 −7.9 retinol dehydrogenase 10(all-trans) 43 GNG4 −7.7 guanine nucleotide binding protein (G protein),gamma 4 44 COP1 −7.4 caspase-1 dominant-negative inhibitor pseudo-ICE 45B3GNT5 −7.4 UDP-GlcNAc:betaGal beta-1,3-N- acetylglucosaminyltransferase5 46 CST7 −7.3 cystatin F (leukocystatin) 47 GLUD2 −7.3 glutamatedehydrogenase 2 48 MCTP1 −7.1 multiple C2 domains, transmembrane 1 49ANXA1 −7.0 annexin A1 50 TBCEL −6.9 tubulin folding cofactor E-like 51CECR1 −6.8 cat eye syndrome chromosome region, candidate 1 52 NIPA1 −6.7non imprinted in Prader- Willi/Angelman syndrome 1 53 FASLG −6.4 Fasligand (TNF superfamily, member 6) 54 MGAT5 −6.4 mannosyl(alpha-1,6-)-glycoprotein beta-1,6-N-acetyl- glucosaminyltransferase 55ACRC −6.4 acidic repeat containing 56 ARHGEF3 −6.3 Rho guaninenucleotide exchange factor (GEF) 3 57 PMCH −6.2pro-melanin-concentrating hormone 58 LPHN2 −6.1 latrophilin 2 59 DPP4−6.0 dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexingprotein 2) 60 FRY −5.9 furry homolog (Drosophila) 61 TA-NFKBH −5.8T-cell activation NFKB-like protein 62 NFIL3 −5.7 nuclear factor,interleukin 3 regulated 63 GPR18 −5.7 G protein-coupled receptor 18 64WDFY1 −5.7 WD repeat and FYVE domain containing 1 65 STAT4 −5.7 signaltransducer and activator of transcription 4 66 C1orf150 −5.7 chromosome1 open reading frame 150 67 TP53INP1 −5.7 tumor protein p53 induciblenuclear protein 1 68 RCAN1 −5.6 regulator of calcineurin 1 69 SRGN −5.6serglycin 70 MTSS1 −5.5 metastasis suppressor 1 71 NR4A2 −5.5 nuclearreceptor subfamily 4, group A, member 2 72 FAM84B −5.4 family withsequence similarity 84, member B 73 FAM107B −5.4 family with sequencesimilarity 107, member B 74 PLK2 −5.4 polo-like kinase 2 (Drosophila) 75GBP2 −5.4 guanylate binding protein 2, interferon- inducible 76 ANK3−5.4 ankyrin 3, node of Ranvier (ankyrin G) 77 EPSTI1 −5.3 epithelialstromal interaction 1 (breast) 78 ENPP2 −5.3 ectonucleotidepyrophosphatase/phos- phodiesterase 2 (autotaxin) 79 SLC16A6 −5.3 solutecarrier family 16, member 6 (monocarboxylic acid transporter 7) 80 GLUD1−5.2 glutamate dehydrogenase 1 81 SERPINB9 −5.2 serpin peptidaseinhibitor, clade B (ovalbumin), member 9 82 NPTX2 −5.2 neuronalpentraxin II 83 CCND1 −5.1 cyclin D1 84 NINJ1 −5.1 ninjurin 1 85 CBLB−5.0 Cas-Br-M (murine) ecotropic retroviral transforming sequence b 86CD200 −5.0 CD200 molecule 87 CHML −5.0 choroideremia-like (Rab escortprotein 2) 88 ZHX2 −5.0 zinc fingers and homeoboxes 2 89 IL22 −4.9interleukin 22 90 FST −4.9 follistatin 91 NR4A3 −4.9 nuclear receptorsubfamily 4, group A, member 3 92 TNFSF11 −4.8 tumor necrosis factor(ligand) superfamily, member 11 93 FYN −4.8 FYN oncogene related to SRC,FGR, YES 94 CCNYL1 −4.7 cyclin Y-like 1 95 CXCR3 −4.7 chemokine (C—X—Cmotif) receptor 3 96 RUNX2 −4.6 runt-related transcription factor 2 97BTLA −4.6 B and T lymphocyte associated 98 CUGBP2 −4.5 CUG tripletrepeat, RNA binding protein 2 99 AGK −4.5 acylglycerol kinase 100LOC650794 −4.5 similar to FRAS1-related extracellular matrix protein 2precursor (ECM3 homolog) 101 IL18RAP −4.5 interleukin 18 receptoraccessory protein 102 MARCKS −4.4 myristoylated alanine-rich proteinkinase C substrate 103 PDE4D −4.4 phosphodiesterase 4D, cAMP-specific(phosphodiesterase E3 dunce homolog, Drosophila) 104 ANKRD10 −4.4ankyrin repeat domain 10 105 IL18R1 −4.4 interleukin 18 receptor 1 106TGFBR3 −4.4 transforming growth factor, beta receptor III 107 SNAG1 −4.4sorting nexin associated golgi protein 1 108 LRRN3 −4.3 leucine richrepeat neuronal 3 109 AFF3 −4.3 AF4/FMR2 family, member 3 110 AXIN2 −4.3axin 2 (conductin, axil) 111 PMCHL1 −4.3 pro-melanin-concentratinghormone- like 1 112 SGPP2 −4.3 sphingosine-1-phosphate phosphotase 2 113SMAD3 −4.3 SMAD family member 3 114 ITGA9 −4.2 integrin, alpha 9 115ABCD3 −4.2 ATP-binding cassette, sub-family D (ALD), member 3 116 MAL−4.2 mal, T-cell differentiation protein 117 IL10 −4.2 interleukin 10118 LOC360030 −4.2 homeobox C14 119 GAD1 −4.2 glutamate decarboxylase 1(brain, 67 kDa) 120 ZCCHC14 −4.2 zinc finger, CCHC domain containing 14121 LOC387763 −4.1 hypothetical LOC387763 122 FOSB −4.1 FBJ murineosteosarcoma viral oncogene homolog B 123 BCL2L1 −4.1 BCL2-like 1 124SPAG1 −4.1 sperm associated antigen 1 125 PHEX −4.0 phosphate regulatingendopeptidase homolog, X-linked (hypophos- phatemia, vitamin D resistantrickets) 126 TAGAP −4.0 T-cell activation GTPase activating protein 127MTX3 −4.0 metaxin 3 128 LAMP3 −4.0 lysosomal-associated membrane protein3 129 SMC5 −4.0 structural maintenance of chromosomes 5 130 CD160 −4.0CD160 molecule 131 PELI1 −4.0 pellino homolog 1 (Drosophila) 132 JAZF1−4.0 JAZF zinc finger 1 133 GBP3 −4.0 guanylate binding protein 3 134 F3−4.0 coagulation factor III (thromboplastin, tissue factor) 135 GEM −4.0GTP binding protein overexpressed in skeletal muscle 136 RNF145 −3.9ring finger protein 145 137 PAM −3.9 peptidylglycine alpha-amidatingmonooxygenase 138 FAM122A −3.9 family with sequence similarity 122A 139C6orf190 −3.9 chromosome 6 open reading frame 190 140 SERAC1 −3.9 serineactive site containing 1 141 FLJ20273 −3.9 RNA-binding protein 142 LAG3−3.9 lymphocyte-activation gene 3 143 FAIM3 −3.8 Fas apoptoticinhibitory molecule 3 144 TBL1X −3.8 transducin (beta)-like 1X-linked145 BCL2L11 −3.8 BCL2-like 11 (apoptosis facilitator) 146 HS3ST3B1 −3.8heparan sulfate (glucosamine) 3-O- sulfotransferase 3B1 147 LOC151162−3.8 hypothetical protein LOC151162 148 ARL5B −3.8 ADP-ribosylationfactor-like 5B 149 BCL2 −3.8 B-cell CLL/lymphoma 2 150 HEG1 −3.8 HEGhomolog 1 (zebrafish) 151 YPEL5 −3.8 yippee-like 5 (Drosophila) 152FRMD4B −3.8 FERM domain containing 4B 153 PARP14 −3.8 poly (ADP-ribose)polymerase family, member 14 154 POU2AF1 −3.8 POU class 2 associatingfactor 1 155 CXorf6 −3.8 chromosome X open reading frame 6 156 C13orf15−3.7 chromosome 13 open reading frame 15 157 NIN −3.7 ninein (GSK3Binteracting protein) 158 ATXN1 −3.7 ataxin 1 159 ATHL1 −3.7 ATH1, acidtrehalase-like 1 (yeast) 160 CRIM1 −3.7 cysteine rich transmembrane BMPregulator 1 (chordin-like) 161 EGLN3 −3.7 egl nine homolog 3 (C.elegans) 162 LBH −3.6 limb bud and heart development homolog (mouse) 163SEMA3D −3.6 sema domain, immunoglobulin domain (Ig), short basic domain,secreted, (semaphorin) 3D 164 MTUS1 −3.6 mitochondrial tumor suppressor1 165 GNAI1 −3.6 guanine nucleotide binding protein (G protein), alphainhibiting activity polypeptide 1 166 PHLDA1 −3.6 pleckstrinhomology-like domain, family A, member 1 167 KLF12 −3.6 Kruppel-likefactor 12 168 PLA2G4A −3.6 phospholipase A2, group IVA (cytosolic,calcium-dependent) 169 DACH1 −3.6 dachshund homolog 1 (Drosophila) 170CD247 −3.5 CD247 molecule 171 RP5-1022P6.2 −3.5 hypothetical proteinKIAA1434 172 MAPKAPK2 −3.5 mitogen-activated protein kinase- activatedprotein kinase 2 173 ATP9A −3.5 ATPase, Class II, type 9A 174 KIAA1913−3.4 KIAA1913 175 PTPRJ −3.4 protein tyrosine phosphatase, receptortype, J 176 ANKRD13C −3.4 ankyrin repeat domain 13C 177 ANKH −3.4ankylosis, progressive homolog (mouse) 178 APOBEC3B −3.3 apolipoproteinB mRNA editing enzyme, catalytic polypeptide-like 3B 179 SIPA1L1 −3.3signal-induced proliferation-associated 1 like 1 180 MGC39606 −3.3hypothetical protein MGC39606 181 C15orf5 −3.3 chromosome 15 openreading frame 5 182 CCL4 −3.3 chemokine (C-C motif) ligand 4 183 CD84−3.3 CD84 molecule 184 CYR61 −3.3 cysteine-rich, angiogenic inducer, 61185 ZNF75 −3.3 zinc finger protein 75 (D8C6) 186 CDKN2C −3.3cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) 187 EGFL6 −3.3EGF-like-domain, multiple 6 188 NCALD −3.3 neurocalcin delta 189 MAP3K5−3.3 mitogen-activated protein kinase kinase kinase 5 190 TRIB2 −3.3tribbles homolog 2 (Drosophila) 191 DLEU2 −3.3 deleted in lymphocyticleukemia, 2 192 TRIB1 −3.3 tribbles homolog 1 (Drosophila) 193 FAM3C−3.3 family with sequence similarity 3, member C 194 REL −3.3 v-relreticuloendotheliosis viral oncogene homolog (avian) 195 TRIM25 −3.3tripartite motif-containing 25 196 GJB6 −3.2 gap junction protein, beta6 197 PLEKHA7 −3.2 pleckstrin homology domain containing, family Amember 7 198 FLJ32810 −3.2 hypothetical protein FLJ32810 199 PHF20L1−3.2 PHD finger protein 20-like 1 200 CYLD −3.1 cylindromatosis (turbantumor syndrome) 201 CPM −3.1 carboxypeptidase M 202 EDARADD −3.1EDAR-associated death domain 203 SRGAP2 −3.1 SLIT-ROBO Rho GTPaseactivating protein 2 204 FBXO30 −3.1 F-box protein 30 205 PKIA −3.1protein kinase (cAMP-dependent, catalytic) inhibitor alpha 206 G0S2 −3.1G0/G1switch 2 207 PRF1 −3.1 perforin 1 (pore forming protein) 208MGC16121 −3.1 hypothetical protein MGC16121 209 SRD5A2L −3.1 steroid 5alpha-reductase 2-like 210 OSBPL3 −3.1 oxysterol binding protein-like 3211 OTUD4 −3.1 OTU domain containing 4 212 IL1RN −3.1 interleukin 1receptor antagonist 213 MB −3.1 myoglobin 214 UQCRC2 −3.0ubiquinol-cytochrome c reductase core protein II 215 CAMK2D −3.0calcium/calmodulin-dependent protein kinase (CaM kinase) II delta 216DAPK1 −3.0 death-associated protein kinase 1 217 KIAA0256 −3.0 KIAA0256gene product 218 SETBP1 −3.0 SET binding protein 1 219 ST8SIA4 −3.0 ST8alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 220 IMAA −3.0SLC7A5 pseudogene 221 CD52 −3.0 CD52 molecule 222 SLAMF6 −3.0 SLAMfamily member 6 223 CTNNA1 −3.0 catenin (cadherin-associated protein),alpha 1, 102 kDa 224 C10orf54 −3.0 chromosome 10 open reading frame 54225 CASP7 −2.9 caspase 7, apoptosis-related cysteine peptidase 226HIVEP1 −2.9 human immunodeficiency virus type I enhancer binding protein1 227 HDGFRP3 −2.9 hepatoma-derived growth factor, related protein 3 228ELF1 −2.9 E74-like factor 1 (ets domain transcription factor) 229 STAT5A−2.9 signal transducer and activator of transcription 5A 230 BBS12 −2.9Bardet-Biedl syndrome 12 231 GTDC1 −2.9 glycosyltransferase-like domaincontaining 1 232 CCDC41 −2.9 coiled-coil domain containing 41 233 ARMCX3−2.9 armadillo repeat containing, X-linked 3 234 STARD4 −2.9StAR-related lipid transfer (START) domain containing 4 235 TSPAN2 −2.9tetraspanin 2 236 MDN1 −2.9 MDN1, midasin homolog (yeast) 237 IRAK2 −2.8interleukin-1 receptor-associated kinase 2 238 TGIF1 −2.8 TGFB-inducedfactor homeobox 1 239 FLJ43663 −2.8 hypothetical protein FLJ43663 240SFXN1 −2.8 sideroflexin 1 241 JMJD3 −2.8 jumonji domain containing 3 242APBB1IP −2.8 amyloid beta (A4) precursor protein- inding, family B,member 1 interacting protein 243 RERE −2.8 arginine-glutamic aciddipeptide (RE) repeats 244 TNFRSF9 −2.8 tumor necrosis factor receptorsuperfamily, member 9 245 RFX2 −2.8 regulatory factor X, 2 (influencesHLA class II expression) 246 SNX9 −2.8 sorting nexin 9 247 CREM −2.8cAMP responsive element modulator 248 SIAH2 −2.8 seven in absentiahomolog 2 (Drosophila) 249 IKZF1 −2.8 IKAROS family zinc finger 1(Ikaros) 250 HTR2B −2.8 5-hydroxytryptamine (serotonin) receptor 2B 251SQLE −2.8 squalene epoxidase 252 GIMAP7 −2.7 GTPase, IMAP family member7 253 C21orf71 −2.7 chromosome 21 open reading frame 71 254 RPS6KA3 −2.7ribosomal protein S6 kinase, 90 kDa, polypeptide 3 255 SYNE2 −2.7spectrin repeat containing, nuclear envelope 2 256 CSF1 −2.7 colonystimulating factor 1 (macrophage) 257 MBNL2 −2.7 muscleblind-like 2(Drosophila) 258 PBEF1 −2.7 pre-B-cell colony enhancing factor 1 259FLJ10038 −2.7 hypothetical protein FLJ10038 260 LAIR2 −2.7leukocyte-associated immunoglobulin- like receptor 2 261 ARHGEF7 −2.7Rho guanine nucleotide exchange factor (GEF) 7 262 XIRP1 −2.7 xinactin-binding repeat containing 1 263 LOC729697 −2.7 hypotheticalprotein LOC729697 264 OGT −2.7 O-linked N-acetylglucosamine (GlcNAc)transferase (UDP-N- acetylglucosamine:polypeptide-N- acetylglucosaminyltransferase) 265 RBM33 −2.7 RNA binding motif protein 33 266 STK4 −2.7serine/threonine kinase 4 267 SOS1 −2.7 son of sevenless homolog 1(Drosophila) 268 AMIGO2 −2.7 adhesion molecule with Ig-like domain 2 269CD99 −2.7 CD99 molecule 270 STAP1 −2.7 signal transducing adaptor familymember 1 271 JARID2 −2.7 jumonji, AT rich interactive domain 2 272KIAA1546 −2.7 KIAA1546 273 RPUSD4 −2.7 RNA pseudouridylate synthasedomain containing 4 274 IL21 −2.7 interleukin 21 275 SAMD9 −2.6 sterilealpha motif domain containing 9 276 FN1 −2.6 fibronectin 1 277 SYTL3−2.6 synaptotagmin-like 3 278 RAB38 −2.6 RAB38, member RAS oncogenefamily 279 DUSP5 −2.6 dual specificity phosphatase 5 280 SORBS1 −2.6sorbin and SH3 domain containing 1 281 SHC4 −2.6 SHC (Src homology 2domain containing) family, member 4 282 ING3 −2.6 inhibitor of growthfamily, member 3 283 KLF4 −2.6 Kruppel-like factor 4 (gut) 284 PRKCH−2.6 protein kinase C, eta 285 C20orf82 −2.6 chromosome 20 open readingframe 82 286 ZC3H12C −2.6 zinc finger CCCH-type containing 12C 287IGF2BP3 −2.6 insulin-like growth factor 2 mRNA binding protein 3 288SAMD4A −2.6 sterile alpha motif domain containing 4A 289 MYLIP −2.6myosin regulatory light chain interacting protein 290 OPA1 −2.6 opticatrophy 1 (autosomal dominant) 291 TRPM6 −2.6 transient receptorpotential cation channel, subfamily M, member 6 292 PTPRK −2.5 proteintyrosine phosphatase, receptor type, K 293 GABPB2 −2.5 GA bindingprotein transcription factor, beta subunit 2 294 FOS −2.5 v-fos FBJmurine osteosarcoma viral oncogene homolog 295 SOX8 −2.5 SRY (sexdetermining region Y)-box 8 296 BIRC3 −2.5 baculoviral IAPrepeat-containing 3 297 CCDC64 −2.5 coiled-coil domain containing 64 298HPSE −2.5 heparanase 299 DENND4A −2.5 DENN/MADD domain containing 4A 300RUNX3 −2.5 runt-related transcription factor 3 301 LOC645431 −2.5hypothetical protein LOC645431 302 CTNS −2.5 cystinosis, nephropathic303 GPR81 −2.5 G protein-coupled receptor 81 304 ATP1B3 −2.5 ATPase,Na+/K+ transporting, beta 3 polypeptide 305 MAPK1 −2.5 mitogen-activatedprotein kinase 1 306 TNFRSF4 −2.5 tumor necrosis factor receptorsuperfamily, member 4 307 PDE3B −2.5 phosphodiesterase 3B,cGMP-inhibited 308 STT3B −2.5 STT3, subunit of theoligosaccharyltransferase complex, homolog B (S. cerevisiae) 309 TP53BP2−2.5 tumor protein p53 binding protein, 2 310 SPRY1 −2.5 sprouty homolog1, antagonist of FGF signaling (Drosophila) 311 EIF4ENIF1 −2.5eukaryotic translation initiation factor 4E nuclear import factor 1 312TLE4 −2.5 transducin-like enhancer of split 4 (E(sp1) homolog,Drosophila) 313 TRAF5 −2.5 TNF receptor-associated factor 5 314 IFNAR2−2.5 interferon (alpha, beta and omega) receptor 2 315 ITPR1 −2.5inositol 1,4,5-triphosphate receptor, type 1 316 KIAA1217 −2.4 KIAA1217317 GZMA −2.4 granzyme A (granzyme 1, cytotoxic T- lymphocyte-associatedserine esterase 3) 318 CD27 −2.4 CD27 molecule 319 PDXDC1 −2.4pyridoxal-dependent decarboxylase domain containing 1 320 SFRS11 −2.4splicing factor, arginine/serine-rich 11 321 BRAF −2.4 v-raf murinesarcoma viral oncogene homolog B1 322 CD80 −2.4 CD80 molecule 323 VPS37B−2.4 vacuolar protein sorting 37 homolog B (S. cerevisiae) 324 FNBP1−2.4 formin binding protein 1 325 FAM113B −2.4 family with sequencesimilarity 113, member B 326 FAM62B −2.4 family with sequence similarity62 (C2 domain containing) member B 327 GPR56 −2.4 G protein-coupledreceptor 56 328 ITGB1 −2.4 integrin, beta 1 (fibronectin receptor, betapolypeptide, antigen CD29 includes MDF2, MSK12) 329 MLSTD1 −2.4 malesterility domain containing 1 330 EGR3 −2.4 early growth response 3 331MITF −2.4 microphthalmia-associated transcription factor 332 SEC61B −2.4Sec61 beta subunit 333 PPP4R2 −2.4 protein phosphatase 4, regulatorysubunit 2 334 BCL10 −2.4 B-cell CLL/lymphoma 10 335 SIPA1L2 −2.4signal-induced proliferation-associated 1 like 2 336 KIAA0182 −2.4KIAA0182 337 USP12 −2.4 ubiquitin specific peptidase 12 338 RNF19A −2.4ring finger protein 19A 339 MAPRE2 −2.4 microtubule-associated protein,RP/EB family, member 2 340 DKFZp547E087 −2.4 hypothetical gene LOC283846341 TSPAN5 −2.4 tetraspanin 5 342 SNF1LK2 −2.4 SNF1-like kinase 2 343MEF2A −2.3 myocyte enhancer factor 2A 344 EGR4 −2.3 early growthresponse 4 345 HSD17B12 −2.3 hydroxysteroid (17-beta) dehydrogenase 12346 C3orf26 −2.3 chromosome 3 open reading frame 26 347 SERPINE2 −2.3serpin peptidase inhibitor, clade E (nexin, plasminogen activatorinhibitor type 1), member 2 348 C10orf18 −2.3 chromosome 10 open readingframe 18 349 FMNL2 −2.3 formin-like 2 350 CHSY1 −2.3 carbohydrate(chondroitin) synthase 1 351 DLEU2L −2.3 deleted in lymphocytic leukemia2-like 352 LOC145474 −2.3 hypothetical protein LOC145474 353 IGF2R −2.3insulin-like growth factor 2 receptor 354 GVIN1 −2.3 GTPase, very largeinterferon inducible 1 355 HOMER2 −2.3 homer homolog 2 (Drosophila) 356ZNF432 −2.3 zinc finger protein 432 357 IL6ST −2.3 interleukin 6 signaltransducer (gp130, oncostatin M receptor) 358 CLEC2D −2.3 C-type lectindomain family 2, member D 359 JAK2 −2.3 Janus kinase 2 (a proteintyrosine kinase) 360 CUGBP1 −2.3 CUG triplet repeat, RNA binding protein1 361 OPN3 −2.3 opsin 3 (encephalopsin, panopsin) 362 CRTAM −2.3cytotoxic and regulatory T cell molecule 363 LYST −2.3 lysosomaltrafficking regulator 364 SLC5A3 −2.3 solute carrier family 5 (inositoltransporters), member 3 365 ANKRD28 −2.3 ankyrin repeat domain 28 366ATP13A3 −2.3 ATPase type 13A3 367 EIF3C −2.3 eukaryotic translationinitiation factor 3, subunit C 368 TNFRSF18 −2.3 tumor necrosis factorreceptor superfamily, member 18 369 REV3L −2.3 REV3-like, catalyticsubunit of DNA polymerase zeta (yeast) 370 RYBP −2.3 RING1 and YY1binding protein 371 FHIT −2.3 fragile histidine triad gene 372 HNRPH1−2.3 heterogeneous nuclear ribonucleoprotein H1 (H) 373 ENC1 −2.3ectodermal-neural cortex (with BTB- like domain) 374 C16orf45 −2.3chromosome 16 open reading frame 45 375 STAT5B −2.3 signal transducerand activator of transcription 5B 376 KIAA1267 −2.3 KIAA1267 377 SDC4−2.3 syndecan 4 378 ITGB7 −2.3 integrin, beta 7 379 PTEN −2.3phosphatase and tensin homolog (mutated in multiple advanced cancers 1)380 BCLAF1 −2.3 BCL2-associated transcription factor 1 381 CD47 −2.3CD47 molecule 382 P2RY5 −2.3 purinergic receptor P2Y, G-protein coupled,5 383 MRPS6 −2.2 mitochondrial ribosomal protein S6 384 ETS1 −2.2 v-etserythroblastosis virus E26 oncogene homolog 1 (avian) 385 IL1R1 −2.2interleukin 1 receptor, type I 386 LRRFIP1 −2.2 leucine rich repeat (inFLII) interacting protein 1 387 C9orf3 −2.2 chromosome 9 open readingframe 3 388 RREB1 −2.2 ras responsive element binding protein 1 389C7orf30 −2.2 chromosome 7 open reading frame 30 390 HMGCS1 −2.23-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) 391 PTPRC−2.2 protein tyrosine phosphatase, receptor type, C 392 RLF −2.2rearranged L-myc fusion 393 CDC14A −2.2 CDC14 cell division cycle 14homolog A (S. cerevisiae) 394 ADAT2 −2.2 adenosine deaminase,tRNA-specific 2, TAD2 homolog (S. cerevisiae) 395 NOL10 −2.2 nucleolarprotein 10 396 TIAM1 −2.2 T-cell lymphoma invasion and metastasis 1 397CD96 −2.2 CD96 molecule 398 SNTB2 −2.2 syntrophin, beta 2 (dystrophin-associated protein A1, 59 kDa, basic component 2) 399 PRKX −2.2 proteinkinase, X-linked 400 DPYD −2.2 dihydropyrimidine dehydrogenase 401PIK3R1 −2.2 phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha)402 LOC23117 −2.2 KIAA0220-like protein 403 DLG1 −2.2 discs, largehomolog 1 (Drosophila) 404 TM2D3 −2.2 TM2 domain containing 3 405 HEATR1−2.2 HEAT repeat containing 1 406 CEACAM21 −2.2 carcinoembryonicantigen-related cell adhesion molecule 21 407 SLC8A1 −2.2 solute carrierfamily 8 (sodium/calcium exchanger), member 1 408 NR4A1 −2.2 nuclearreceptor subfamily 4, group A, member 1 409 SRP54 −2.2 signalrecognition particle 54 kDa 410 TULP4 −2.2 tubby like protein 4 411ZFP36L1 −2.2 zinc finger protein 36, C3H type-like 1 412 C20orf74 −2.2chromosome 20 open reading frame 74 413 CENTD1 −2.2 centaurin, delta 1414 NSUN4 −2.2 NOL1/NOP2/Sun domain family, member 4 415 AZIN1 −2.2antizyme inhibitor 1 416 PCSK6 −2.2 proprotein convertasesubtilisin/kexin type 6 417 PDS5A −2.2 PDS5, regulator of cohesionmaintenance, homolog A (S. cerevisiae) 418 DDHD1 −2.2 DDHD domaincontaining 1 419 LTV1 −2.2 LTV1 homolog (S. cerevisiae) 420 KIF1B −2.2kinesin family member 1B 421 EFNB2 −2.2 ephrin-B2 422 HBP1 −2.2 HMG-boxtranscription factor 1 423 SLTM −2.2 SAFB-like, transcription modulator424 TMEM88 −2.2 transmembrane protein 88 425 PHACTR2 −2.2 phosphataseand actin regulator 2 426 SPATA13 −2.2 spermatogenesis associated 13 427NHS −2.2 Nance-Horan syndrome (congenital cataracts and dentalanomalies) 428 FOXN3 −2.2 forkhead box N3 429 EOMES −2.2 eomesoderminhomolog (Xenopus laevis) 430 WNK1 −2.2 WNK lysine deficient proteinkinase 1 431 HS3ST1 −2.2 heparan sulfate (glucosamine) 3-O-sulfotransferase 1 432 SBF2 −2.2 SET binding factor 2 433 TWIST1 −2.2twist homolog 1 (acrocephalosyndactyly 3; Saethre- Chotzen syndrome)(Drosophila) 434 RASGRF2 −2.2 Ras protein-specific guaninenucleotide-releasing factor 2 435 ATF6 −2.2 activating transcriptionfactor 6 436 SF1 −2.2 splicing factor 1 437 TMEM133 −2.2 transmembraneprotein 133 438 CABIN1 −2.2 calcineurin binding protein 1 439 CHST11−2.2 carbohydrate (chondroitin 4) sulfotransferase 11 440 LGALS3BP −2.2lectin, galactoside-binding, soluble, 3 binding protein 441 ZNF419 −2.2zinc finger protein 419 442 CNOT2 −2.1 CCR4-NOT transcription complex,subunit 2 443 LOC643187 −2.1 similar to ankyrin repeat domain 20A 444PRKCB1 −2.1 protein kinase C, beta 1 445 KBTBD6 −2.1 kelch repeat andBTB (POZ) domain containing 6 446 POLR3C −2.1 polymerase (RNA) III (DNAdirected) polypeptide C (62 kD) 447 C4orf32 −2.1 chromosome 4 openreading frame 32 448 TRIM13 −2.1 tripartite motif-containing 13 449TMEM165 −2.1 transmembrane protein 165 450 C1orf104 −2.1 chromosome 1open reading frame 104 451 FOSL2 −2.1 FOS-like antigen 2 452 LOC650392−2.1 hypothetical protein LOC650392 453 PQLC1 −2.1 PQ loop repeatcontaining 1 454 PLCL1 −2.1 phospholipase C-like 1 455 WTAP −2.1 Wilmstumor 1 associated protein 456 SMU1 −2.1 smu-1 suppressor of mec-8 andunc-52 homolog (C. elegans) 457 KCNQ5 −2.1 potassium voltage-gatedchannel, KQT-like subfamily, member 5 458 ANP32A −2.1 acidic(leucine-rich) nuclear phos- phoprotein 32 family, member A 459 MUC20−2.1 mucin 20, cell surface associated 460 LOC152485 −2.1 hypotheticalprotein LOC152485 461 NETO1 −2.1 neuropilin (NRP) and tolloid (TLL)-like 1 462 LOC440345 −2.1 hypothetical protein LOC440345 463 ZNF567 −2.1zinc finger protein 567 464 SUV420H1 −2.1 suppressor of variegation 4-20homolog 1 (Drosophila) 465 MGC7036 −2.1 hypothetical protein MGC7036 466WIPI1 −2.1 WD repeat domain, phosphoinositide interacting 1 467 PER1−2.1 period homolog 1 (Drosophila) 468 TNFRSF21 −2.1 tumor necrosisfactor receptor superfamily, member 21 469 RBM25 −2.1 RNA binding motifprotein 25 470 EIF2C2 −2.1 eukaryotic translation initiation factor 2C,2 471 NPAS3 −2.1 neuronal PAS domain protein 3 472 KIAA0922 −2.1KIAA0922 473 TRIM6 −2.1 tripartite motif-containing 6 474 LOC440993 −2.1hypothetical gene supported by AK128346 475 PIM3 −2.1 pim-3 oncogene 476MBNL1 −2.1 muscleblind-like (Drosophila) 477 LOC730092 −2.1 RRN3 RNApolymerase I transcription factor homolog (S. cerevisiae) pseudogene 478CYSLTR1 −2.1 cysteinyl leukotriene receptor 1 479 RAB8B −2.1 RAB8B,member RAS oncogene family 480 UBE2F −2.1 ubiquitin-conjugating enzymeE2F (putative) 481 PRDM1 −2.1 PR domain containing 1, with ZNF domain482 ADK −2.1 adenosine kinase 483 LOC727738 −2.1 similar to Amphiregulinprecursor (AR) (Colorectum cell-derived growth factor) (CRDGF) 484 PEX3−2.1 peroxisomal biogenesis factor 3 485 CD55 −2.1 CD55 molecule, decayaccelerating factor for complement (Cromer blood group) 486 AUTS2 −2.1autism susceptibility candidate 2 487 MAP3K8 −2.1 mitogen-activatedprotein kinase kinase kinase 8 488 ESR2 −2.1 estrogen receptor 2 (ERbeta) 489 UBE2B −2.1 ubiquitin-conjugating enzyme E2B (RAD6 homolog) 490SNX8 −2.1 sorting nexin 8 491 RICTOR −2.1 rapamycin-insensitivecompanion of mTOR 492 CD7 −2.1 CD7 molecule 493 IQGAP2 −2.1 IQ motifcontaining GTPase activating protein 2 494 ANKRD44 −2.1 ankyrin repeatdomain 44 495 MGC12916 −2.1 hypothetical protein MGC12916 496 TM6SF1−2.1 transmembrane 6 superfamily member 1 497 GPR171 −2.1 Gprotein-coupled receptor 171 498 NR1D2 −2.1 nuclear receptor subfamily1, group D, member 2 499 HLA-E −2.1 major histocompatibility complex,class I, E 500 ZFAND3 −2.1 zinc finger, AN1-type domain 3 501 OAS3 −2.12′-5′-oligoadenylate synthetase 3, 100 kDa 502 IFIT5 −2.1interferon-induced protein with tetratricopeptide repeats 5 503 SLC16A14−2.1 solute carrier family 16, member 14 (monocarboxylic acidtransporter 14) 504 PFAAP5 −2.1 phosphonoformate immuno-associatedprotein 5 505 SNRK −2.1 SNF related kinase 506 PYHIN1 −2.1 pyrin and HINdomain family, member 1 507 GAN −2.1 giant axonal neuropathy (gigaxonin)508 KIAA0265 −2.1 KIAA0265 protein 509 PCBP2 −2.1 poly(rC) bindingprotein 2 510 PLGLB1 −2.0 plasminogen-like B1 511 NASP −2.0 nuclearautoantigenic sperm protein (histone-binding) 512 MIRH1 −2.0 microRNAhost gene (non-protein coding) 1 513 CD44 −2.0 CD44 molecule (Indianblood group) 514 MAP2K3 −2.0 mitogen-activated protein kinase kinase 3515 TRPS1 −2.0 trichorhinophalangeal syndrome I 516 EPB41L4A −2.0erythrocyte membrane protein band 4.1 like 4A 517 MGEA5 −2.0 meningiomaexpressed antigen 5 (hyaluronidase) 518 CTSS −2.0 cathepsin S 519 TBC1D4−2.0 TBC1 domain family, member 4 520 UFM1 −2.0 ubiquitin-fold modifier1 521 RAB11FIP1 −2.0 RAB11 family interacting protein 1 (class I) 522RAPH1 −2.0 Ras association (RaIGDS/AF-6) and pleckstrin homology domains1 523 PRPF39 −2.0 PRP39 pre-mRNA processing factor 39 homolog (S.cerevisiae) 524 HIPK1 −2.0 homeodomain interacting protein kinase 1 525NR3C1 −2.0 nuclear receptor subfamily 3, group C, member 1(glucocorticoid receptor) 526 ZNF148 −2.0 zinc finger protein 148 527RPS27 −2.0 ribosomal protein S27 (metallopanstimulin 1) 528 MARCH6 −2.0membrane-associated ring finger (C3HC4) 6 529 SNX5 −2.0 sorting nexin 5530 IL32 −2.0 interleukin 32 531 ZBTB11 −2.0 zinc finger and BTB domaincontaining 11 532 TBCA −2.0 tubulin folding cofactor A 533 C16orf52 −2.0chromosome 16 open reading frame 52 534 UBE2E1 −2.0ubiquitin-conjugating enzyme E2E 1 (UBC4/5 homolog, yeast) 535 ETV6 −2.0ets variant gene 6 (TEL oncogene) 536 C1orf9 −2.0 chromosome 1 openreading frame 9 537 PPP1R15A −2.0 protein phosphatase 1, regulatory(inhibitor) subunit 15A 538 SFRS4 −2.0 splicing factor,arginine/serine-rich 4 539 ZNF26 −2.0 zinc finger protein 26 540 ZNF814−2.0 zinc finger protein 814 541 CUL4A −2.0 cullin 4A 542 TMEM2 −2.0transmembrane protein 2 543 COPA −2.0 coatomer protein complex, subunitalpha 544 SLC35F5 −2.0 solute carrier family 35, member F5 545 AKAP11−2.0 A kinase (PRKA) anchor protein 11 546 SETD2 −2.0 SET domaincontaining 2 547 SLC7A1 −2.0 solute carrier family 7 (cationic aminoacid transporter, y+ system), member 1 548 YME1L1 −2.0 YME1-like 1 (S.cerevisiae) 549 EIF2AK3 −2.0 eukaryotic translation initiation factor2-alpha kinase 3 550 TCERG1 −2.0 transcription elongation regulator 1

While certain of the particular embodiments of the present inventionhave been described and specifically exemplified above, it is notintended that the invention be limited to such embodiments. Variousmodifications may be made thereto without departing from the scope andspirit of the present invention, as set forth in the following claims.

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SEQUENCE INFORMATION

Genbank Accession No.: NM_005060 The following is the human RORgammamRNA sequence. The nucleotides unique for RORgamma are underlined.

>gi|48255916: 109-1665 Homo sapiens RAR-relatedorphan receptor C (RORC), transcript variant 1, mRNA (SEQ ID NO: 29)ATGGACAGGGCCCCACAGAGACAGCACCGAGCCTCACGGGAGCTGCTGGCTGCAAAGAAGACCCACACCTCACAAATTGAAGTGATCCCTTGCAAAATCTGTGGGGACAAGTCGTCTGGGATCCACTACGGGGTTATCACCTGTGAGGGGTGCAAGGGCTTCTTCCGCCGGAGCCAGCGCTGTAACGCGGCCTACTCCTGCACCCGTCAGCAGAACTGCCCCATCGACCGCACCAGCCGAAACCGATGCCAGCACTGCCGCCTGCAGAAATGCCTGGCGCTGGGCATGTCCCGAGATGCTGTCAAGTTCGGCCGCATGTCCAAGAAGCAGAGGGACAGCCTGCATGCAGAAGTGCAGAAACAGCTGCAGCAGCGGCAACAGCAGCAACAGGAACCAGTGGTCAAGACCCCTCCAGCAGGGGCCCAAGGAGCAGATACCCTCACCTACACCTTGGGGCTCCCAGACGGGCAGCTGCCCCTGGGCTCCTCGCCTGACCTGCCTGAGGCTTCTGCCTGTCCCCCTGGCCTCCTGAAAGCCTCAGGCTCTGGGCCCTCATATTCCAACAACTTGGCCAAGGCAGGGCTCAATGGGGCCTCATGCCACCTTGAATACAGCCCTGAGCGGGGCAAGGCTGAGGGCAGAGAGAGCTTCTATAGCACAGGCAGCCAGCTGACCCCTGACCGATGTGGACTTCGTTTTGAGGAACACAGGCATCCTGGGCTTGGGGAACTGGGACAGGGCCCAGACAGCTACGGCAGCCCCAGTTTCCGCAGCACACCGGAGGCACCCTATGCCTCCCTGACAGAGATAGAGCACCTGGTGCAGAGCGTCTGCAAGTCCTACAGGGAGACATGCCAGCTGCGGCTGGAGGACCTGCTGCGGCAGCGCTCCAACATCTTCTCCCGGGAGGAAGTGACTGGCTACCAGAGGAAGTCCATGTGGGAGATGTGGGAACGGTGTGCCCACCACCTCACCGAGGCCATTCAGTACGTGGTGGAGTTCGCCAAGAGGCTCTCAGGCTTTATGGAGCTCTGCCAGAATGACCAGATTGTGCTTCTCAAAGCAGGAGCAATGGAAGTGGTGCTGGTTAGGATGTGCCGGGCCTACAATGCTGACAACCGCACGGTCTTTTTTGAAGGCAAATACGGTGGCATGGAGCTGTTCCGAGCCTTGGGCTGCAGCGAGCTCATCAGCTCCATCTTTGACTTCTCCCACTCCCTAAGTGCCTTGCACTTTTCCGAGGATGAGATTGCCCTCTACACAGCCCTTGTTCTCATCAATGCCCATCGGCCAGGGCTCCAAGAGAAAAGGAAAGTAGAACAGCTGCAGTACAATCTGGAGCTGGCCTTTCATCATCATCTCTGCAAGACTCATCGCCAAAGCATCCTGGCAAAGCTGCCACCCAAGGGGAAGCTTCGGAGCCTGTGTAGCCAGCATGTGGAAAGGCTGCAGATCTTCCAGCACCTCCACCCCATCGTGGTCCAAGCCGCTTTCCCTCCACTCTACAAGGAGCTCTTCAGCACTGAAACCGAGTCACCTGTGGGGCTGTCCAAGTGA

Genbank Accession No.: NP_005051 The following is the human RORgammaprotein sequence. The amino acids unique for RORgamma are underlined.The AF2 domain is indicated using underlining and bold font.

>gi|19743909|ref|NP_005051.2| RAR-related orphanreceptor C isoform a [Homo sapiens] (SEQ ID NO: 30)MDRAPQRQHRASRELLAAKKTHTSQIEVIPCKICGDKSSGIHYGVITCEGCKGFFRRSQRCNAAYSCTRQQNCPIDRTSRNRCQHCRLQKCLALGMSRDAVKFGRMSKKQRDSLHAEVQKQLQQRQQQQQEPVVKTPPAGAQGADTLTYTLGLPDGQLPLGSSPDLPEASACPPGLLKASGSGPSYSNNLAKAGLNGASCHLEYSPERGKAEGRESFYSTGSQLTPDRCGLRFEEHRHPGLGELGQGPDSYGSPSFRSTPEAPYASLTEIEHLVQSVCKSYRETCQLRLEDLLRQRSNIFSREEVTGYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTVFFEGKYGGMELFRALGCSELISSIFDFSHSLSALHFSEDEIALYTALVLINAHRPGLQEKRKVEQLQYNLELAFHHHLCKTHRQSILAKLPPKGKLRSLCSQHVERLQIFQHLHPIVVQAAFPP LYKELF STETESPVGLSK

Genbank Accession No.: NM_001001523 The following is the human RORgammatmRNA sequence. The nucleotides unique for RORgamma are underlined.

>gi|48255917: 142-1635 Homo sapiens RAR-relatedorphan receptor C (RORC), transcript variant 2, mRNA (SEQ ID NO: 31)ATGAGAACACAAATTGAAGTGATCCCTTGCAAAATCTGTGGGGACAAGTCGTCTGGGATCCACTACGGGGTTATCACCTGTGAGGGGTGCAAGGGCTTCTTCCGCCGGAGCCAGCGCTGTAACGCGGCCTACTCCTGCACCCGTCAGCAGAACTGCCCCATCGACCGCACCAGCCGAAACCGATGCCAGCACTGCCGCCTGCAGAAATGCCTGGCGCTGGGCATGTCCCGAGATGCTGTCAAGTTCGGCCGCATGTCCAAGAAGCAGAGGGACAGCCTGCATGCAGAAGTGCAGAAACAGCTGCAGCAGCGGCAACAGCAGCAACAGGAACCAGTGGTCAAGACCCCTCCAGCAGGGGCCCAAGGAGCAGATACCCTCACCTACACCTTGGGGCTCCCAGACGGGCAGCTGCCCCTGGGCTCCTCGCCTGACCTGCCTGAGGCTTCTGCCTGTCCCCCTGGCCTCCTGAAAGCCTCAGGCTCTGGGCCCTCATATTCCAACAACTTGGCCAAGGCAGGGCTCAATGGGGCCTCATGCCACCTTGAATACAGCCCTGAGCGGGGCAAGGCTGAGGGCAGAGAGAGCTTCTATAGCACAGGCAGCCAGCTGACCCCTGACCGATGTGGACTTCGTTTTGAGGAACACAGGCATCCTGGGCTTGGGGAACTGGGACAGGGCCCAGACAGCTACGGCAGCCCCAGTTTCCGCAGCACACCGGAGGCACCCTATGCCTCCCTGACAGAGATAGAGCACCTGGTGCAGAGCGTCTGCAAGTCCTACAGGGAGACATGCCAGCTGCGGCTGGAGGACCTGCTGCGGCAGCGCTCCAACATCTTCTCCCGGGAGGAAGTGACTGGCTACCAGAGGAAGTCCATGTGGGAGATGTGGGAACGGTGTGCCCACCACCTCACCGAGGCCATTCAGTACGTGGTGGAGTTCGCCAAGAGGCTCTCAGGCTTTATGGAGCTCTGCCAGAATGACCAGATTGTGCTTCTCAAAGCAGGAGCAATGGAAGTGGTGCTGGTTAGGATGTGCCGGGCCTACAATGCTGACAACCGCACGGTCTTTTTTGAAGGCAAATACGGTGGCATGGAGCTGTTCCGAGCCTTGGGCTGCAGCGAGCTCATCAGCTCCATCTTTGACTTCTCCCACTCCCTAAGTGCCTTGCACTTTTCCGAGGATGAGATTGCCCTCTACACAGCCCTTGTTCTCATCAATGCCCATCGGCCAGGGCTCCAAGAGAAAAGGAAAGTAGAACAGCTGCAGTACAATCTGGAGCTGGCCTTTCATCATCATCTCTGCAAGACTCATCGCCAAAGCATCCTGGCAAAGCTGCCACCCAAGGGGAAGCTTCGGAGCCTGTGTAGCCAGCATGTGGAAAGGCTGCAGATCTTCCAGCACCTCCACCCCATCGTGGTCCAAGCCGCTTTCCCTCCACTCTACAAGGAGCTCTTCAGCACTGAAACCGAGTCACCTGTGGGGCTGTCCAAGTGA

Genbank Accession No.: NP_001001523 The following is the human RORgammatprotein sequence. The amino acids unique for RORgammat are underlined.The AF2 domain is indicated using underlining and bold font.

>gi|48255918|ref|NP_001001523.1| RAR-related orphanreceptor C isoform b [Homo sapiens] (SEQ ID NO: 32)MRTQIEVIPCKICGDKSSGIHYGVITCEGCKGFFRRSQRCNAAYSCTRQQNCPIDRTSRNRCQHCRLQKCLALGMSRDAVKFGRMSKKQRDSLHAEVQKQLQQRQQQQQEPVVKTPPAGAQGADTLTYTLGLPDGQLPLGSSPDLPEASACPPGLLKASGSGPSYSNNLAKAGLNGASCHLEYSPERGKAEGRESFYSTGSQLTPDRCGLRFEEHRHPGLGELGQGPDSYGSPSFRSTPEAPYASLTEIEHLVQSVCKSYRETCQLRLEDLLRQRSNIFSREEVTGYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTVFFEGKYGGMELFRALGCSELISSIFDFSHSLSALHFSEDEIALYTALVLINAHRPGLQEKRKVEQLQYNLELAFHHHLCKTHRQSILAKLPPKGKLRSLCSQHVERLQIFQHLHPIVVQAAFPP LYKELF STETESPVGLSK

Genbank Accession No.: NM_011281 The following is the mRNA sequence formouse RORgamma. The underlined sequences are specific for mouseRORgamma.

>gi|6755343: 107-1657 Mus musculus RAR-relatedorphan receptor gamma (Rorc), mRNA (SEQ ID NO: 33)ATGGACAGGGCCCCACAGAGACACCACCGGACATCTCGGGAGCTGCTGGCTGCAAAGAAGACCCACACCTCACAAATTGAAGTGATCCCTTGCAAGATCTGTGGGGACAAGTCATCTGGGATCCACTACGGGGTTATCACCTGTGAGGGGTGCAAGGGCTTCTTCCGCCGCAGCCAGCAGTGTAATGTGGCCTACTCCTGCACGCGTCAGCAGAACTGCCCCATTGACCGAACCAGCCGCAACCGATGCCAGCATTGCCGCCTGCAGAAGTGCCTGGCTCTGGGCATGTCCCGAGATGCTGTCAAGTTTGGCCGAATGTCCAAGAAGCAGAGGGACAGTCTACATGCAGAAGTGCAGAAACAACTGCAACAGCAGCAGCAACAGGAACAAGTGGCCAAGACTCCTCCAGCTGGGAGCCGCGGAGCAGACACACTTACATACACTTTAGGGCTCTCAGATGGGCAGCTACCACTGGGCGCCTCACCTGACCTACCCGAGGCCTCTGCTTGTCCCCCTGGCCTCCTGAGAGCCTCAGGCTCTGGCCCACCATATTCCAATACCTTGGCCAAAACAGAGGTCCAGGGGGCCTCCTGCCACCTTGAGTATAGTCCAGAACGAGGCAAAGCTGAAGGCAGAGACAGCATCTATAGCACTGACGGCCAACTTACTCTTGGAAGATGTGGACTTCGTTTTGAGGAAACCAGGCATCCTGAACTTGGGGAACCAGAACAGGGTCCAGACAGCCACTGCATTCCCAGTTTCTGCAGTGCCCCAGAGGTACCATATGCCTCTCTGACAGACATAGAGTACCTGGTACAGAATGTCTGCAAGTCCTTCCGAGAGACATGCCAGCTGCGACTGGAGGACCTTCTACGGCAGCGCACCAACCTCTTTTCACGGGAGGAGGTGACCAGCTACCAGAGGAAGTCAATGTGGGAGATGTGGGAGCGCTGTGCCCACCACCTCACTGAGGCCATTCAGTATGTGGTGGAGTTTGCCAAGCGGCTTTCAGGCTTCATGGAGCTCTGCCAGAATGACCAGATCATACTACTGACAGCAGGAGCAATGGAAGTCGTCCTAGTCAGAATGTGCAGGGCCTACAATGCCAACAACCACACAGTCTTTTTTGAAGGCAAATACGGTGGTGTGGAGCTGTTTCGAGCCTTGGGCTGCAGCGAGCTCATCAGCTCCATATTTGACTTTTCCCACTTCCTCAGCGCCCTGTGTTTTTCTGAGGATGAGATTGCCCTCTACACGGCCCTGGTTCTCATCAATGCCAACCGTCCTGGGCTCCAAGAGAAGAGGAGAGTGGAACATCTGCAATACAATTTGGAACTGGCTTTCCATCATCATCTCTGCAAGACTCATCGACAAGGCCTCCTAGCCAAGCTGCCACCCAAAGGAAAACTCCGGAGCCTGTGCAGCCAACATGTGGAAAAGCTGCAGATCTTCCAGCACCTCCACCCCATCGTGGTCCAAGCCGCCTTCCCGCCACTCTATAAGGAACTCTTCAGCACTGATGTTGAATCCCCTGAGGGGCTGTCAAAGTG A

Genbank Accession No.: NP_035411 The following is the amino acidsequence for mouse RORgamma. The underlined amino acids are specific formouse RORgamma. The AF2 domain is indicated using underlining and boldfont.

>gi|6755344|ref|NP_035411.1| RAR-related orphanreceptor gamma [Mus musculus] (SEQ ID NO: 34)MDRAPQRHHRTSRELLAAKKTHTSQIEVIPCKICGDKSSGIHYGVITCEGCKGFFRRSQQCNVAYSCTRQQNCPIDRTSRNRCQHCRLQKCLALGMSRDAVKFGRMSKKQRDSLHAEVQKQLQQQQQQEQVAKTPPAGSRGADTLTYTLGLSDGQLPLGASPDLPEASACPPGLLRASGSGPPYSNTLAKTEVQGASCHLEYSPERGKAEGRDSIYSTDGQLTLGRCGLRFEETRHPELGEPEQGPDSHCIPSFCSAPEVPYASLTDIEYLVQNVCKSFRETCQLRLEDLLRQRTNLFSREEVTSYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIILLTAGAMEVVLVRMCRAYNANNHTVFFEGKYGGVELFRALGCSELISSIFDFSHFLSALCFSEDEIALYTALVLINANRPGLQEKRRVEHLQYNLELAFHHHLCKTHRQGLLAKLPPKGKLRSLCSQHVEKLQIFQHLHPIVVQAAFPP LY KELF STDVESPEGLSK

Genbank Accession No.: AF163668 The following is the mRNA sequence formouse RORgammat. The underlined nucleotides are specific for mouseRORgammat

>gi|5679306: 93-1580 Mus musculus RORgamma t mRNA, complete cds(SEQ ID NO: 35) ATGAGAACACAAATTGAAGTGATCCCTTGCAAGATCTGTGGGGACAAGTCATCTGGGATCCACTACGGGGTTATCACCTGTGAGGGGTGCAAGGGCTTCTTCCGCCGCAGCCAGCAGTGTAATGTGGCCTACTCCTGCACGCGTCAGCAGAACTGCCCCATTGACCGAACCAGCCGCAACCGATGCCAGCATTGCCGCCTGCAGAAGTGCCTGGCTCTGGGCATGTCCCGAGATGCTGTCAAGTTTGGCCGAATGTCCAAGAAGCAGAGGGACAGTCTACATGCAGAAGTGCAGAAACAACTGCAACAGCAGCAGCAACAGGAACAAGTGGCCAAGACTCCTCCAGCTGGGAGCCGCGGAGCAGACACACTTACATACACTTTAGGGCTCTCAGATGGGCAGCTACCACTGGGCGCCTCACCTGACCTACCCGAGGCCTCTGCTTGTCCCCCTGGCCTCCTGAGAGCCTCAGGCTCTGGCCCACCATATTCCAATACCTTGGCCAAAACAGAGGTCCAGGGGGCCTCCTGCCACCTTGAGTATAGTCCAGAACGAGGCAAAGCTGAAGGCAGAGACAGCATCTATAGCACTGACGGCCAACTTACTCTTGGAAGATGTGGACTTCGTTTTGAGGAAACCAGGCATCCTGAACTTGGGGAACCAGAACAGGGTCCAGACAGCCACTGCATTCCCAGTTTCTGCAGTGCCCCAGAGGTACCATATGCCTCTCTGACAGACATAGAGTACCTGGTACAGAATGTCTGCAAGTCCTTCCGAGAGACATGCCAGCTGCGACTGGAGGACCTTCTACGGCAGCGCACCAACCTCTTTTCACGGGAGGAGGTGACCAGCTACCAGAGGAAGTCAATGTGGGAGATGTGGGAGCGCTGTGCCCACCACCTCACTGAGGCCATTCAGTATGTGGTGGAGTTTGCCAAGCGGCTTTCAGGCTTCATGGAGCTCTGCCAGAATGACCAGATCATACTACTGAAAGCAGGAGCAATGGAAGTCGTCCTAGTCAGAATGTGCAGGGCCTACAATGCCAACAACCACACAGTCTTTTTTGAAGGCAAATACGGTGGTGTGGAGCTGTTTCGAGCCTTGGGCTGCAGCGAGCTCATCAGCTCCATATTTGACTTTTCCCACTTCCTCAGCGCCCTGTGTTTTTCTGAGGATGAGATTGCCCTCTACACGGCCCTGGTTCTCATCAATGCCAACCGTCCTGGGCTCCAAGAGAAGAGGAGAGTGGAACATCTGCAATACAATTTGGAACTGGCTTTCCATCATCATCTCTGCAAGACTCATCGACAAGGCCTCCTAGCCAAGCTGCCACCCAAAGGAAAACTCCGGAGCCTGTGCAGCCAACATGTGGAAAAGCTGCAGATCTTCCAGCACCTCCACCCCATCGTGGTCCAAGCCGCCTTCCCTCCACTCTATAAGGAACTCTTCAGCACTGATGTTGAATCCCCTGAGGGGCTGTCAAAGTGA

Genbank Accession No.: AAD46913 The following is the amino acid sequencefor mouse RORgammat. The underlined amino acids are specific for mouseRORgammat. The AF2 domain is indicated using underlining and bold font.

>gi|5679307|gb|AAD46913.1|AF163668_1 RORgamma t [Mus musculus](SEQ ID NO: 36) MRTQIEVIPCKICGDKSSGIHYGVITCEGCKGFFRRSQQCNVAYSCTRQQNCPIDRTSRNRCQHCRLQKCLALGMSRDAVKFGRMSKKQRDSLHAEVQKQLQQQQQQEQVAKTPPAGSRGADTLTYTLGLSDGQLPLGASPDLPEASACPPGLLRASGSGPPYSNTLAKTEVQGASCHLEYSPERGKAEGRDSIYSTDGQLTLGRCGLRFEETRHPELGEPEQGPDSHCIPSFCSAPEVPYASLTDIEYLVQNVCKSFRETCQLRLEDLLRQRTNLFSREEVTSYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIILLKAGAMEVVLVRMCRAYNANNHTVFFEGKYGGVELFRALGCSELISSIFDFSHFLSALCFSEDEIALYTALVLINANRPGLQEKRRVEHLQYNLELAFHHHLCKTHRQGLLAKLPPKGKLRSLCSQHVEKLQIFQHLHPIVVQAAFPP LYKELF STDVESPEGLSK

Genbank Accession No.: NM_134262.1 The following is the mRNA sequencefor human RORalpha isoform d.

>gi|19743904|ref|NM_134262.1| Homo sapiens RAR-related orphan receptor A (RORA), transcript variant 4, mRNA(SEQ ID NO: 37) TGTGGCTCGGGCGGCGGCGGCGCGGCGGCGGCAGAGGGGGCTCCGGGGTCGGACCATCCGCTCTCCCTGCGCTCTCCGCACCGCGCTTAAATGATGTATTTTGTGATCGCAGCGATGAAAGCTCAAATTGAAATTATTCCATGCAAGATCTGTGGAGACAAATCATCAGGAATCCATTATGGTGTCATTACATGTGAAGGCTGCAAGGGCTTTTTCAGGAGAAGTCAGCAAAGCAATGCCACCTACTCCTGTCCTCGTCAGAAGAACTGTTTGATTGATCGAACCAGTAGAAACCGCTGCCAACACTGTCGATTACAGAAATGCCTTGCCGTAGGGATGTCTCGAGATGCTGTAAAATTTGGCCGAATGTCAAAAAAGCAGAGAGACAGCTTGTATGCAGAAGTACAGAAACACCGGATGCAGCAGCAGCAGCGCGACCACCAGCAGCAGCCTGGAGAGGCTGAGCCGCTGACGCCCACCTACAACATCTCGGCCAACGGGCTGACGGAACTTCACGACGACCTCAGTAACTACATTGACGGGCACACCCCTGAGGGGAGTAAGGCAGACTCCGCCGTCAGCAGCTTCTACCTGGACATACAGCCTTCCCCAGACCAGTCAGGTCTTGATATCAATGGAATCAAACCAGAACCAATATGTGACTACACACCAGCATCAGGCTTCTTTCCCTACTGTTCGTTCACCAACGGCGAGACTTCCCCAACTGTGTCCATGGCAGAATTAGAACACCTTGCACAGAATATATCTAAATCGCATCTGGAAACCTGCCAATACTTGAGAGAAGAGCTCCAGCAGATAACGTGGCAGACCTTTTTACAGGAAGAAATTGAGAACTATCAAAACAAGCAGCGGGAGGTGATGTGGCAATTGTGTGCCATCAAAATTACAGAAGCTATACAGTATGTGGTGGAGTTTGCCAAACGCATTGATGGATTTATGGAACTGTGTCAAAATGATCAAATTGTGCTTCTAAAAGCAGGTTCTCTAGAGGTGGTGTTTATCAGAATGTGCCGTGCCTTTGACTCTCAGAACAACACCGTGTACTTTGATGGGAAGTATGCCAGCCCCGACGTCTTCAATGAAAAACAAACAAAAAAATTAACCGAGACACTTTATATGGCCCTGCACAGACCTGGAGCGCCACACACTATCCTTAGGTTGTGAAGACTTTATTAGCTTTGTGTTTGAATTTGGAAAGAGTTTATGTTCTATGCACCTGACTGAAGATGAAATTGCATTATTTTCTGCATTTGTACTGATGTCAGCAGATCGCTCATGGCTGCAAGAAAAGGTAAAAATTGAAAAACTGCAACAGAAAATTCAGCTAGCTCTTCAACACGTCCTACAGAAGAATCACCGAGAAGATGGAATACTAACAAAGTTAATATGCAAGGTGTCTACATTAAGAGCCTTATGTGGACGACATACAGAAAAGCTAATGGCATTTAAAGCAATATACCCAGACATTGTGCGACTTCATTTTCCTCCATTATACAAGGAGTTGTTCACTTCAGAATTTGAGCCAGCAATGCAAATTGATGGGTAAATGTTATCACCTAAGCACTTCTAGAATGTCTGAAGTACAAACAGCACATCTTTTGGTGATCGGGGTCAGGCAAAGGAGGGGAAACAATGAAAACAAATAAAGTTGAACTTGTTTTTCTCA

Genbank Accession No.: NP_599024.1 The following is the amino acidsequence for human RORalpha isoform d.

>gi|19743905|ref|NP_599024.1| RAR-related orphanreceptor A isoform d [Homo sapiens] (SEQ ID NO: 38)MMYFVIAAMKAQIEIIPCKICGDKSSGIHYGVITCEGCKGFFRRSQQSNATYSCPRQKNCLIDRTSRNRCQHCRLQKCLAVGMSRDAVKFGRMSKKQRDSLYAEVQKHRMQQQQRDHQQQPGEAEPLTPTYNISANGLTELHDDLSNYIDGHTPEGSKADSAVSSFYLDIQPSPDQSGLDINGIKPEPICDYTPASGFFPYCSFTNGETSPTVSMAELEHLAQNISKSHLETCQYLREELQQITWQTFLQEEIENYQNKQREVMWQLCAIKITEAIQYVVEFAKRIDGFMELCQNDQIVLLKAGSLEVVFIRMCRAFDSQNNTVYFDGKYASPDVFKSLGCEDFISFVFEFGKSLCSMHLTEDEIALFSAFVLMSADRSWLQEKVKIEKLQQKIQLALQHVLQKNHREDGILTKLICKVSTLRALCGRHTEKLMAFKAIYPDIVRLHFPP LYKELFTSEFEPAMQIDG

Genbank Accession No.: NM_006914.3 The following is the mRNA sequencefor human RORbeta.

>gi|62865658|ref|NM_006914.3| Homo sapiens RAR-related orphan receptor B (RORB), mRNA (SEQ ID NO: 39)TCTCTCCCCTCTCTTTCTCTCTCGCTGCTCCCTTCCTCCCTGTAACTGAACAGTGAAAATTCACATTGTGGATCCGCTAACAGGCACAGATGTCATGTGAAAACGCACATGCTCTGCCATCCACACCGCCTTTCTTTCTTTTCTTTCTGTTTCCTTTTTTCCCCCTTGTTCCTTCTCCCTCTTCTTTGTAACTAACAAAACCACCACCAACTCCTCCTCCTGCTGCTGCCCTTCCTCCTCCTCCTCAGTCCAAGTGATCACAAAAGAAATCTTCTGAGCCGGAGGCGGTGGCATTTTTTAAAAAGCAAGCACATTGGAGAGAAAGAAAAAGAAAAACAAAACCAAAACAAAACCCAGGCACCAGACAGCCAGAACATTTTTTTTTCACCCTTCCTGAAAACAAACAAACAAACAAACAATCATCAAAACAGTCACCACCAACATCAAAACTGTTAACATAGCGGCGGCGGCGGCAAACGTCACCCTGCAGCCACGGCGTCCGCCTAAAGGGATGGTTTTCTCGGCAGAGCAGCTCTTCGCCGACCACCTTCTTCACTCGTGCTGAGCGGGATTTTTGGGCTCTCCGGGGTTCGGGCTGGGAGCAGCTTCATGACTACGCGGAGCGGGAGAGCGGCCACACCATGCGAGCACAAATTGAAGTGATACCATGCAAAATTTGTGGCGATAAGTCCTCTGGGATCCACTACGGAGTCATCACATGTGAAGGCTGCAAGGGATTCTTTAGGAGGAGCCAGCAGAACAATGCTTCTTATTCCTGCCCAAGGCAGAGAAACTGTTTAATTGACAGAACGAACAGAAACCGTTGCCAACACTGCCGACTGCAGAAGTGTCTTGCCCTAGGAATGTCAAGAGATGCTGTGAAGTTTGGGAGGATGTCCAAGAAGCAAAGGGACAGCCTGTATGCTGAGGTGCAGAAGCACCAGCAGCGGCTGCAGGAACAGCGGCAGCAGCAGAGTGGGGAGGCAGAAGCCCTTGCCAGGGTGTACAGCAGCAGCATTAGCAACGGCCTGAGCAACCTGAACAACGAGACCAGCGGCACTTATGCCAACGGGCACGTCATTGACCTGCCCAAGTCTGAGGGTTATTACAACGTCGATTCCGGTCAGCCGTCCCCTGATCAGTCAGGACTTGACATGACTGGAATCAAACAGATAAAGCAAGAACCTATCTATGACCTCACATCCGTACCCAACTTGTTTACCTATAGCTCTTTCAACAATGGGCAGTTAGCACCAGGGATAACCATGACTGAAATCGACCGAATTGCACAGAACATCATTAAGTCCCATTTGGAGACATGTCAATACACCATGGAAGAGCTGCACCAGCTGGCGTGGCAGACCCACACCTATGAAGAAATTAAAGCATATCAAAGCAAGTCCAGGGAAGCACTGTGGCAACAATGTGCCATCCAGATCACTCACGCCATCCAATACGTGGTGGAGTTTGCAAAGCGGATAACAGGCTTCATGGAGCTCTGTCAAAATGATCAAATTCTACTTCTGAAGTCAGGTTGCTTGGAAGTGGTTTTAGTGAGAATGTGCCGTGCCTTCAACCCATTAAACAACACTGTTCTGTTTGAAGGAAAATATGGAGGAATGCAAATGTTCAAAGCCTTAGGTTCTGATGACCTAGTGAATGAAGCATTTGACTTTGCAAAGAATTTGTGTTCCTTGCAGCTGACCGAGGAGGAGATCGCTTTGTTCTCATCTGCTGTTCTGATATCTCCAGACCGAGCCTGGCTTATAGAACCAAGGAAAGTCCAGAAGCTTCAGGAAAAAATTTATTTTGCACTTCAACATGTGATTCAGAAGAATCACCTGGATGATGAGACCTTGGCAAAGTTAATAGCCAAGATACCAACCATCACGGCAGTTTGCAACTTGCACGGGGAGAAGCTGCAGGTATTTAAGCAATCTCATCCAGAGATAGTGAATACACTGTTTCCTCCGTTATACAAGGAGCTCTTTAATCCTGACTGTGCCACCGGCTGCAAATGAAGGGGACAAGAGAACTGTCTCATAGTCATGGAATGCATCACCATTAAGACAAAAGCAATGTGTTCATGAAGACTTAAGAAAAATGTCACTACTGCAACATTAGGAATGTCCTGCACTTAATAGAATTATTTTTCACCGCTACAGTTTGAAGAATGTAAATATGCACCTGAGTGGGGCTCTTTTATTTGTTTGTTTGTTTTTGAAATGACCATAAATATACAAATATAGGACACTGGGTGTTATCCTTTTTTTAATTTTATTCGGGTATGTTTTGGGAGACAACTGTTTATAGAATTTTATTGTAGATATATACAAGAAAAGAGCGGTACTTTACATGATTACTTTTCCTGTTGATTGTTCAAATATAATTTAAGAAAATTCCACTTAATAGGCTTACCTATTTCTATGTTTTTAGGTAGTTGATGCATGTGTAAATTTGTAGCTGTCTTGGAAAGTACTGTGCATGTATGTAATAAGTATATAATATGTGAGAATATTATATATGACTATTACTTATACATGCACATGCACTGTGGCTTAAATACCATACCTACTAGCAATGGAGGTTCAGTCAGGCTCTCTTCTATGATTTACCTTCTGTGTTATATGTTACCTTTATGTTAGACAATCAGGATTTTGTTTTCCCAGCCAGAGTTTTCATCTATAGTCAATGGCAGGACGGTACCAACTCAGAGTTAAGTCTACAAAGGAATAAACATAATGTGTGGCCTCTATATACAAACTCTATTTCTGTCAATGACATCAAAGCCTTGTCAAGATGGTTCATATTGGGAAGGAGACAGTATTTTAAGCCATTTTCCTGTTTCAAGAATTAGGCCACAGATAACATTGCAAGGTCCAAGACTTTTTTGACCAAACAGTAGATATTTTCTATTTTTCACCAGAACACATAAAAACACTTTTTTTCTTTTGGATTTCTGGTTGTGAAACAAGCTTGATTTCAGTGCTTATTGTGTCTTCAACTGAAAAATACAATCTGTGGATTATGACTACCAGCAATTTTTTTCTAGGAAAGTTAAAAGAATAAATCAGAACCCAGGGCAACAATGCCATTTCATGTAAACATTTTCTCTCTCACCATGTTTTGGCAAGAAAAGGTAGAAAGAGAAGACCCAGAGTGAAGAAGTAATTCTTTATATTCCTTTCTTTAATGTATTTGTTAGGAAAAGTGGCAATAAAGGGGGAGGCATATTATAAAATGCTATAATATAAAAATGTAGCAAAAACTTGACAGACTAGAAAAAAAAAGATCTGTGTTATTCTAGGGAACTAATGTACCCCAAAGCCAAAACTAATTCCTGTGAAGTTTACAGTTACATCATCCATTTACCCTAGAATTATTTTTTTAGCAACTTTTAGAAATAAAGAATACAACTGTGACATTAGGATCAGAGATTTTAGACTTCCTTGTACAAATTCTCACTTCTCCACCTGCTCACCAATGAAATTAATCATAAGAAAAGCATATATTCCAAGAAATTTGTTCTGCCTGTGTCCTGGAGGCCTATACCTCTGTTATTTTCTGATACAAAATAAAACTTAAAAAAAAGAAAACAAG CTAA

Genbank Accession No.: NP_008845.2 The following is the amino acidsequence for human RORbeta.

>gi|19743907|ref|NP_008845.2| RAR-related orphanreceptor B [Homo sapiens] (SEQ ID NO: 40)MRAQIEVIPCKICGDKSSGIHYGVITCEGCKGFFRRSQQNNASYSCPRQRNCLIDRTNRNRCQHCRLQKCLALGMSRDAVKFGRMSKKQRDSLYAEVQKHQQRLQEQRQQQSGEAEALARVYSSSISNGLSNLNNETSGTYANGHVIDLPKSEGYYNVDSGQPSPDQSGLDMTGIKQIKQEPIYDLTSVPNLFTYSSFNNGQLAPGITMTEIDRIAQNIIKSHLETCQYTMEELHQLAWQTHTYEEIKAYQSKSREALWQQCAIQITHAIQYVVEFAKRITGFMELCQNDQILLLKSGCLEVVLVRMCRAFNPLNNTVLFEGKYGGMQMFKALGSDDLVNEAFDFAKNLCSLQLTEEEIALFSSAVLISPDRAWLIEPRKVQKLQEKIYFALQHVIQKNHLDDETLAKLIAKIPTITAVCNLHGEKLQVFKQSHPEIVNTLFPPLYKELF NPDCATGCK

What is claimed is:
 1. A purified, homogeneous population of antigennaive human Th-IL17+ cells, wherein the purified, homogeneous populationcomprises about or at least 10⁹ antigen naive human Th-IL17+ cells andthe purified, homogeneous population of antigen naive human Th-IL17+cells does not include Th-IL17+ cells expressing cellular markers ofTh1, Th2, or Treg cells.
 2. The purified, homogeneous population ofantigen naive human Th-IL 17+ cells of claim 1, wherein the antigennaive human Th-IL17+ cells express at least one marker of human Th-IL17+cells in addition to IL-17, wherein the at least one marker is IL-17F,IL-26, or RORC.
 3. The purified, homogeneous population of antigen naivehuman Th-IL17+ cells of claim 1, wherein the antigen naive humanTh-IL17+ cells express at least one of the following cell surfacemarkers or cell surface antigens comprising CCR6, IL23R, CD45RO+, CCR7,CCR5, or CXCR4.
 4. The purified, homogeneous population of antigen naivehuman Th-IL17+ cells of claim 1, wherein the antigen naive humanTh-IL17+ cells do not express IFNγ, T-bet, IL4, IL13, GATA-3, or FoxP3.5. A composition comprising the purified, homogeneous population ofantigen naive human Th-IL17+ cells of claim 1 and a pharmaceuticallyacceptable carrier.
 6. The composition of claim 5, further comprisingrecombinant TGF-β.
 7. A composition comprising a purified, homogeneouspopulation of antigen naive human Th-IL17+ cells, wherein the purified,homogeneous population of antigen naive human Th-IL17+ cells does notinclude Th-IL17+ cells expressing cellular markers of Th1, Th2, or Tregcells; recombinant TGF-β; and serum-free culture medium.
 8. Thecomposition of claim 7, wherein the composition further comprises IL-1β,and any one of IL-6, IL-21 or IL-23.
 9. The A purified, homogeneouspopulation of antigen naive human Th-IL17+ cells of claim 1, wherein thepurified, homogeneous population of antigen naive human Th-IL17+ cellsis detectably labeled.
 10. A composition comprising a purifiedhomogeneous population of antigen naive human Th-IL17+ cells, whereinthe purified, homogeneous population of antigen naive human Th-IL17+cells does not include Th-IL17+ cells expressing cellular markers ofTh1, Th2, or Treg cells, generated by a method comprising the steps of:isolating a population of antigen naive CD4+T cells from a human,wherein the antigen naive CD4+T cells are isolated from cord blood,buffy coats of adult humans, cell cultures comprising cells that expressCD34 (CD34+ cells), or human embryonic stem cells; and incubating thepopulation of antigen naive CD4+T cells in serum-free culture mediumcomprising TGF-β, IL-1β, and any one of IL-6, IL-21 or IL-23, whereinthe incubating promotes differentiation of human Th-IL17+ cells.
 11. Apurified, homogeneous population of antigen naive human Th-IL17+ cells,wherein the purified, homogeneous population comprises about or at least10⁹ antigen naive human Th-IL17+ cells.
 12. A composition comprising thepurified, homogeneous population of antigen naive human Th-IL17+ cellsof claim 11 and a pharmaceutically acceptable carrier.